The Astbury Centre for Structural Molecular ANNUAL REPORT 2016 Front cover illustration Folding of the Im7 in the presence or absence of the small, periplasmic ATP-independent chaperone Spy from E. coli. Im7 is shown as a multi-coloured protein that is helical in both the folding intermediate (I) and in the native folded state (N) and lacks any persistent secondary structure in the unfolded state (U). Spy is shown as a blue cradle-shaped homodimer. The kinetic mechanism of Im7 folding in the presence of Spy was investigated as part of a collaboration between the Radford (Leeds) and Bardwell (Michigan) groups and published in Nat. Struct. Mol. Biol. in 2016. More details can be found on p58 of this report.

Acknowledgement The Astbury Centre for Structural Molecular Biology thanks its many sponsors for support of the work and its members for writing these reports. The report is edited by David Brockwell. This report is also available electronically at www.astbury.leeds.ac.uk

I prefix Mission Statement TheAstbury Centre forStructural Molecular Biologywillpromoteinterdisciplinaryresearchofthe higheststandardonthe structure andfunctionofbiological , biomolecularassemblies andcomplexes usingphysico-chemical,molecularbiological andcomputational approaches.

www.astbury.leeds.ac.uk II prefix Introduction Welcometothe Annual Report of theAstbury Centre for2016. We have hadanother very busy andsuccessful year andthisletterhighlights afew of ourmanysuccesses.The Astbury Report is ashining exampleofthe buoyancy of ourscience, thestrengthofour community andour collaborations that go hand in hand with thecontributionsour members make to others through engagementinpublic-facingevents. Iwould liketothank every member of theCentre fortheir hard work over theyear: ourSupport staff, Technicians, Facility Managers, Students, Post-docs, Fellowsand Academic staff. And, of course,LucyGrayfor wonderful organisation andher excellent administrative support.Our successcomes fromour strong multidisciplinary science, as well as ourcollegialityand teamwork.Thank you all!

During2016the Centre continued in its questof“UnderstandingLifeinMolecularDetail” through multipledifferentactivities, includingseminars, publications, public lectures andother events.Wecontinued to enjoyanexcellent seminarseries(organised by JoeCockburn), hosting10lectures during theyearwithspeakersfromthe UK andEurope.Awonderful tenth Annual AstburyLecture wasgiven by SirVenki Ramakrishnan,FRS,(MRC Laboratory of MolecularBiology)onthe 24th June, entitled “The use of recentadvances in electron microscopy to studyribosome structure".Thisannualevent wastraditionally followed by (a rainy!)sportsday andamuch enjoyed barbecue hosted by theAstbury Society (see http://www.astbury.leeds.ac.uk/about/society.php). 139members attended theCentre’s Biennial Away Day, held on September16th at TheThackrayMedical Museum where students,post-docs andPIs shared their recent excitingscientificdiscoveries, through talks, posters andthe ever-popular “Flash Poster”presentations.

Aparticularhighlight of theyearwas launching thefirst ever AstburyConversationthatwas held on 11th &12thApril2016. More than 250eminent researchers from aroundthe world attendedthe AstburyConversation–thefirst internationalevent of itskindatthe University dedicated to thesubject of structuralmolecularbiology.The two-dayevent beganwithan academicresearchsymposium whichwas followedbyapublic exhibitionshowcasing the research andtechnologiesusedinthe University’s AstburyCentre forStructural Molecular Biology. Thefinal highlight wasalecture by NobelLaureate Professor Michael Levitt, FRS, during whichheexplained howcomputer modellinghas advanced ourknowledge of the molecularprocesses underpinninglife, anddisease andwhatthe futureholds forthis area of science. Youcan watch hislectureagain on YouTube (https://www.youtube.com/watch?v=wtQY9IfBqPc)and see http://www.astburyconversation.leeds.ac.uk/ forphotosofthe Conversationevent itself. He also talked abouthow hiscareerdeveloped fromachildhood in SouthAfrica,through research in theUK, Israel andthe USA,tohis NobelPrize in 2013.The AstburyConversationwill run biennially andplans arenow underway forthe 2018 AstburyConversationthatwillbeheld on 16th &17th April 2018 –pleaseadd thesedates to your diary nowand we look forward to seeingyou there!

TheCentrewelcomed Richard Bayliss, JuanFontana, Elton Zeqiraj, Glyn Hemsworthand MeganWrighttothe membershipin2016. As aresultofgenerousfundingfromLeeds alumnus, PeterCheney,wewelcomed visits fromProfessors Carol Hall (NorthCarolinaState University,USA),HerbertWaldman(MaxPlanck, Germany) andHarryTakagi(NIH, USA)and several events/activitieswere held to providethe opportunityfor Fellows andAstbury members to meet andplancollaborations.Wehavereceived fundingfor three more Cheney Fellows in 2017 andlookforward to welcomingKelly ChibaleUniversity of Cape Town,(South Africa),Steve Polyak (University of Washington,Seattle, USA) andPrebenMorth(University of Oslo,Norway) (see http://www.astbury.leeds.ac.uk/people/fellows.php). We were delighted to welcome many PhD students andpostdocstothe Centre this year,bringing ourtotal numbersto>300,including69academicstaff, 208PhD students,93postdoctoral researchers and9Research Fellows.

III prefix

AstburyCentre members publishedtheir research in awiderange of journals in 2016 anda full listcan be foundatthe endofthisreport. In termsofgrantincome,Astbury membersalso enjoyedmanysuccessesin2016. Togetherwith£9.3M of newprojectand programme grants, this brings theAstbury grantportfolio to astriking£52Mshare of £85M of grants: an impressive figure that is testamenttothe hard work andsuccess of ourmembers.Some of thelarger awards included a£1.25MEPSRC EstablishedCareer FellowshiptoAdamNelson;a£1.5M Cancer Research UK Programmegrant to Richard Baylissand Wellcome Trustinvestigator award (£1.8M)toSheenaRadford.LarsJeuken, Michael McPherson andDarren Tomlinson were part of asuccessfulUniversity of Leedsconsortiumbid (“Accelerating Developmentof InfectionDiagnostics forPatient Management andReductionofAntibioticMisuse”(£3.8M) fromthe MRC-coordinated AMR2 initiative. We are much indebted to thefunding agencies that support ourscience, includingBBSRC,EPSRC,MRC,the WellcomeTrust,charities, ERC, EU andIndustry. We also acknowledge, withthanks, thesupport of theUniversity of Leeds; theFaculties of Biological Sciences andMaths andPhysical Sciences andthe Schools of ,Molecularand CellularBiology,Biomedical Sciences andPhysicsand Astronomyfor theirsupport of theCentreand ourresearch.

Therewas continued success in 2016 forseveralmembers of theAstbury Centre in termsof peer recognition. Alison Ashcroft wasawarded -Membershipofthe BritishMass SpectrometrySocietyand Andy Wilson wasannounced as the2016recipient of theRSC Norman Heatley Award forthe developmentofmethods to interrogate andmanipulate protein- proteininteractionsusing biomimetic approaches.Our students andpost-docs also were awardedmanyprizes fortheir contributionsatconferences andmeetings that spanthe globe: EthanMorgan(Wellcome Trustfunded)was awarded thirdplace in thenationalYoung Microbiologistofthe Year competition; Emma Pool (Zhougroup) wonthe best poster pricefor herposteratthe RSC Northern Dalton Meeting, PatrickKnight(BBSRC DTPstudent)won a poster prizeatthe 2016 BMSS meeting, Sarah Hewitt(Wilson group)was awarded aposter prizeatthe MSMLGmeeting in Bath forher work on rutheniumbased protein surface mimetics.Welldoneall.

TheAstbury Society,led by thepresident Matt JacksonplayedaspectacularroleinAstbury activities in 2016.Eventsincludedthe famous Christmasquiznight,and ahugelysuccessful fourth MayBall. With continuedfundraisingthrough cake bakesand coffee mornings, the Society continuestosupport the“LeedsChildren’sCharity”withdonations reaching an impressive £2884.15 to date.

Ihopethatyou enjoyreading this Annual Reportand its newlook. Thankyou to David Brockwelland Lucy Gray forediting this report,everyonewho contributedtoit, andall who participated in theAstbury Centre’s activities in 2016. Ilookforward to continuingour successes in theyearahead.

Sheena E. Radford,FMedSci,FRS

Director, AstburyCentrefor Structural MolecularBiology, Leeds, March 2016

Please notethatthisreport(as well as thosefromprevious years) is also availableasaPDF document whichcan be downloaded from ourwebsite (www.astbury.leeds.ac.uk).

www.astbury.leeds.ac.uk IV prefix CONTENTS

Pages Efficiency of trapping by -Harvestingproteins 1 Peter Adams Biomolecularmassspectrometry andstructuralproteomics 2-3 JamesAult, SamuelBunce,Antonio Calabrese, Owen Cornwell, Paul Devine, Rachel George,Kate Groves, PatrickKnight, Esther Martin, TomWatkinson, Leon Willis,Lydia Youngand Alison Ashcroft Host-cellinteractions of RiskGroup4viruses 4-5 Rebecca Surtees, Amelia Shaw,Jamel Mankouri,ThomasEdwards and JohnBarr Themany(interacting)faces of Aurora-A kinase 6-7 Selena Burgess, Mark Richardsand Richard Bayliss Bio-inspired model membranes and light-harvestingnanomaterials 8-9 Sanobar Khan,MengqiuLi, StephenMuench, Lars Jeuken andPaul Beales Proteinengineeringofaldolases 10-11 ClaireWindle,RobertSmith,AlexMoloney, Naim Stiti, Adam Nelson andAlanBerry Engineeringnovel naturalproductsand bindingmotifs 12-13 Ieva Drulyte, EmilyTurri,AdamNelson andAlanBerry Economicaland scalable synthesis of 6-amino-2-cyanobenzothiazole 14 JacobHauser, Hester Beard, Stuart Warriner andRobinBon Structuralstudiesofauto-inhibitionand mutationalactivationinfibroblast 15 growth-factorreceptors BrendanFarrell,GaryThompson,ArnoutKalverdaand Alex Breeze Structuralbiology of themicrotubule cytoskeleton 16 JoeCockburn Thephysics of life in extreme environments 17-18 KasiaTych,Matthew Batchelor, Toni Hoffmann, Michael Wilson,Megan Hughes,SamuelLenton,DanielleWalsh,NatashaRhys,JamesTowey, Emanuele Paci,DavidBrockwelland LornaDougan Ferroptosisasanew celldeath pathwayinvolved in neurodegeneration 19-20 Andrew Tsatsanis,Bruce Wong andJames Duce Structure-based design of inhibitorsofmetallo E-lactamases: rescuing our 21-22 currentantibiotics Ricky Cain and Colin Fishwick Newsmallmolecule inhibitors of T. gondii bc1:towards acurefor 23-24 toxoplasmosis Martin McPhillie,James Gordon,Stephen Muench, andColinFishwick Understanding howthe fusion protein of Herpes SimplexVirus-1,gB, drives 25 fusion betweenthe virusand thehostcellmembranes Lauren Brownand JuanFontana Developmentofanovelsmall anticoagulant with minimalbleeding 26 risk RogerTaylor, ColinFishwick, CharlotteRevill, Emma Hethershaw, Richard Foster Structuraland functional studiesofmembranepyrophosphatases 27-28 CraigWilkinson, NitaShah, Steven Harborne andAdrian Goldman Causativeand curativeviruses in humancancer 29-30 AbigailBloy, MatthewBentham,ClaireScott,Hannah Beaumont,Emma Brown, ElizabethAppleton, Mohamed Madkour, Adel Samsonand StephenGriffin

V prefix Fluctuating Finite ElementAnalysis-asimulationpackage forbiomolecular 31-32 simulationofelectronmicroscopy database(EMDB) structures AlbertSolernou, BenHanson, GlennCarrington,Rob Welchand Sarah Harris Towards abetter understanding of oxidativebiomass deconstruction 33 Glyn Hemsworth Production, purification andcharacterizationofproteins of theproteobacterial 34-35 acinetobacterchlorhexidineefflux, PACEfamily involved in antimicrobial resistance IrshadAhmad, Karl Hassan,Scott Jackson, IanPaulsen andPeter Henderson An integrated approach to thestudy of cellularinteractions with amyloid 36 MatthewJackson,Clare Pashley, , Eric Hewitt Amodel membrane system to mimicfoldedcristae of mitochondriaand 37-38 thylakoidstacksofchloroplast George Heath, MengqiuLi, LarsJeuken Optimalreactioncoordinates 39-40 PolinaBanushkina andSergeiKrivov Receptor tyrosine kinase signallinginthe absence of growth factorstimulation 41-42 EleanorCawthorne,Janne Darell, Christopher Jones, Sabine Knapp, Chi-ChuanLin,Dovile Milonaityte, ArndtRohwedder,CarolineSeiler, and JohnLadbury Understanding therequirement forion channel activity during virusinfection 43 SamanthaHover, BeckyFoster, Eleanor Todd,JohnBarr and Jamel Mankouri An improved binary cloningsystemfor studying themechanisticbiology of 44-45 Michael Watson, Yu-fei Lin, Elizabeth Hollwey, Rachel Dodds, Peter Meyerand KennethMcDowall Electron microscopy of membrane to underpin structure guided 46-47 inhibitordesign ShaunRawson, Emily Caseley, Paul Beales, Lin-HuaJiang,Colin Fishwickand StephenMuench Unifiedsyntheticapproachestoexplore biologically-relevantchemical space 48-49 IgnacioColomer,Philip Craven, Richard Doveston, Christopher Empson, James Firth, DanielFoley, MatthewLilburn,ZachOwenand Adam Nelson ABC-F proteins mediateantibioticresistance throughribosomal protection 50-51 Liam Sharkey, Thomas Edwardsand Alex O’Neill Transcellularstress signalling: howproteotoxicstress is communicated 52-53 betweendifferent tissuesinC.elegans Daniel O’Brien, Rebecca Aston, VijayShanmugiah,DavidWestheadand Patricija van Oosten-Hawle Proteins underthe computational :folding,disorder, mechanics, 54-55 dynamics andself-assembly Emanuele Paci Howmyosin10travels to filopodiatips 56-57 Ruth Hughes,MarcinWolny,Matthew Batchelor, Francine Parker, BrendanRogers, Glen Carrington,Marta Kurzawa,Tom Baboolaland Michelle Peckham Insights into thefoldingmechanismsofperiplasmicchaperones 58-59 BobSchiffrin, Julia Humes, Anton Calabrese, Paul Devine,Sarah Harris,Alison Ashcroft, DavidBrockwell andSheenaRadford Themoleculardetails of amyloid fibril-glycosaminoglycan binding 60-61 Katie Stewartand Sheena Radford

www.astbury.leeds.ac.uk VI prefix /DWHUDO RSHQLQJLQWKH LQWDFW ȕ-barrelassembly machinerycaptured by cryo- 62-63 EM Anna Higgins, MatthewIadanza,Bob Schiffrin, AntonioCalabrese, Alison Ashcroft, DavidBrockwell, Sheena Radfordand Neil Ranson Coordinate regulationofantimycin andcandicidinbiosynthesis 64 ThomasMcLeanand Ryan Seipke StudyofsensoryneuronsofC.elegans usingamicrofluidic device 65 Jinyang Chungand Jung-ukShim Simvastatin andfluvastatin interactwithhuman gapjunctiongamma-3 protein 66 Paul Taylor Positive allosteric modulationofTRPV1 using Affimer reagents 67-68 RobBedford,MikeMcPherson andDarrenTomlinson Regulationofmuscle contraction by titinand myosinbindingproteinC 69 CharlotteScarff,AlbaFuentes Balaguer, MehrnazMontazeri,Larissa Tskhovrebova andJohn Trinick Cooperativemechanismofprotein translocation through Sec machinery 70-71 Peter Oatley, TomasFessl,StephenBaldwin,Sheena Radfordand Roman Tuma Theroleofstructured RNAinearly virusreplicationevents 72 Andrew Tuplin Novelantiviral strategies forhuman herpesviruses 73-74 Sophie Schumann, BelindaBaquero-Perez, Charlotte Revill, Richard Foster andAdrianWhitehouse Designed inhibitors of protein-proteininteractions 75-76 George Burslem,ClaireGrison,Sarah Hewitt,Katherine Horner, Thomas James, Hannah Kyle,JenniferMiles, ChrisPask, Silvia Rodriguez-Marin, PhilipRowell, AlexBreeze, ThomasEdwards, Adam Nelson,Stuart Warriner, Andrew Wilson Profilingprotein lipidation in parasiteswithchemical tools 77-78 MeganWright Probingmultivalent protein-carbohydrateinteractions using compact, 79-80 polyvalent glycan-quantum dotand Försterresonanceenergy transfer Yuan Guo, Chadamas Sakonsinsiri,EmmaPool, WeiliWang, W. Bruce Turnbulland DejianZhou

VII prefix Contributions indexedbyAstburyCentrePrincipal Investigator

Adams……… 1 Ashcroft ………2,58,60 Barr ………4,43 Bayliss……… 6 Beales……… 8,46 Berry……… 10,12 Bon……… 14 Breeze ………15,75 Brockwell……… 17,58,60 Cockburn ………16 Dougan ………17 Duce ………19 Edwards ………4,50,75 Fishwick……… 21,23,46 Fontana ………25 Foster ………26,73 Goldman ……… 27 Griffin ………29 Harris,S.……… 31,60 Hemsworth……… 33 Henderson ………34 Hewitt ………36 Jeuken……… 8,37 Krivov ………39 Ladbury ………41 Mankouri ………43 McDowall ………44 Muench ………8,23,46 Nelson ………10,12,48,75 O’Neill ………50 vOosten-Hawle……… 52 Paci ……… 17,54 Peckham……… 56 Radford……… 36,58,60,62,70 Ranson ……… 62 Seipke ……… 64 Shim ……… 65 Taylor ……… 66 Tomlinson……… 67 Trinick……… 69 Tuma ……… 70 Tuplin ……… 72 Warriner ……… 14,75 Westhead ……… 52 Whitehouse ……… 73 Wilson ……… 75 Wright M……… 77 Zhou ………79

www.astbury.leeds.ac.uk VIII main Efficiencyofenergy trappingbyLight-Harvestingproteins

Peter Adams

Introduction We understand much aboutthe fundamentals of photosynthesisfrommodel ,such as purple phototrophic bacteria (e.g. Rhodobacter species) includingthe structure,function andorganisationoftheir LH membranes. In naturalphotosyntheticmembranes,anantenna system of light-harvesting(LH)membrane protein complexes absorb photonsvia embedded pigmentcofactors andchannel that energy downhilltothe Reaction Center (RC) whereenergy trappingoccursvia photochemical chargeseparation.InRhodobactersphaeroides,there are twotypes LH complex:(i) the‘core complex’, comprisedofLight-Harvestingcomplex 1(LH1) encirclingthe RC and(ii)the peripheral Light-Harvestingcomplex2(LH2). Thecorecomplex structure is quitevariable betweendifferentspecies and it is stillnot understood howthis relates to theefficiency of energy trapping by theRC. Thenativedimericformofcore complexes is foundinwild-type strains, comprised of twoRCsubunitssurrounded by an S- shapedarrangement of 28 LH1subunits(Fig. 1, upper, blue). Whereas,amonomericformof core complexes, comprisedofonlyone RC enclosed by aring-shaped enclosure of 15 LH1 subunitsisfound in PufX-truncation mutant strains(Fig. 1, lower, red).

Results We investigated theeffect of core complexstructure on itsenergytrappingbyinvestigationof aseriesofdifferent compositionsofphotosynthetic membranesusing various spectroscopies andanalyses. Membranes containing either (i)dimericor(ii)monomeric core complexes and amatched content of LH2antenna complexes were extracted.LHproteincontent was measured by absorptionspectroscopy andgraphical analysis. Time-resolved fluorescence spectroscopy (Fig.1,right)revealed that sampleswithmonomeric coreshaveconsistently longer thefluorescencelifetimethanthose of dimeric cores, irrespective of theirLH2 content or thestate of theRC(active or saturated). Akinetic modelofexciton transferproduced good agreementbetween experimental andcalculated fluorescenceyield.Thiscorrelates with the findingofahigher quantumefficiency forenergytrappingincore complexdimers.Wepropose that theshorterlifetime fordimeric complexesisdue to excitationsharing across alargerLH1 antenna with access to twotraps (RCs), resultinginincreased efficiency of trappingorquenching LH1 excitons.

Publications ChenchiliyanM., Timpmann K.,Jalviste E.,Adams P.G.,HunterC.N.&Freiberg A. (2016) Dimerizationofcore complexes as an efficientstrategyfor energy trapping in Rhodobactersphaeroides. Biochim. Biophys. Acta-Bioenerg. 1857:634-642.

Funding Figure 1: schematic (left): Energy transferin This work wasfunded by theBBSRC. photosynthetic vesiclescontaining either dimeric(blue) or monomeric(red) corecomplexes and LH2(green). Collaborators Graphs (right):Picosecondtime-resolved fluorescence External: C. Hunter (University of spectra fromcorrespondingsamples (samecolour- coding).Fluorescencelifetime fitisdisplayedinpsnext Sheffield), A. Freiberg (University of Tartu, to the corresponding curve. Estonia).

1 main Biomolecular mass spectrometry andstructural proteomics

James Ault,SamuelBunce, AntonioCalabrese,OwenCornwell, Paul Devine,Rachel George,KateGroves, PatrickKnight, Esther Martin,Tom Watkinson, Leon Willis, Lydia Youngand AlisonAshcroft

Introduction Ourresearch is focussedonthe developmentand applicationofmassspectrometry (MS) to investigate thestructure andfunctionofbiomolecules. We usenon-covalentelectrospray ionisation (ESI)-MS,tandemmassspectrometry(MS/MS) andion mobilityspectrometry (IMS)-MStodetermine themass, conformational properties, stoichiometry, stability, and bindingcharacteristicsofbiomolecules andtheir complexes. Specifically,westudy protein folding, function andself-aggregation, protein-ligandinteractionsand biomolecularcomplex assembly.Wealsouse chemical labelling methodsinconjunction with ESI-MSand ESI- MS/MS to mapprotein foldingand aggregationpathwaysand to characterisenon-covalently boundbiomolecularcomplexes (Figure1).

Figure 1: chemical labelling methods used with MS for structural proteomics to study protein structure and identify protein:protein andprotein:ligand interactions include: hydrogen- deuteriumexchange (HDX), hydroxyl radical foot-printing (FPOP) and chemical crosslinking.

Results Ourmajor projectsare aimedatcharacterising amyloidprotein aggregationand inhibition,for whichwehavedevelopedahigh-throughputscreening method to evaluate potentialsmall molecule amyloidinhibitors, determiningmembrane proteinstructure andfunction(Figure 2), anddevelopingnew biomolecularMSmethodologies.

Figure 2: theouter membrane proteinOmpTisshown (green ribbon), highlighting itsposition with respecttothe trans- andextra-membrane regions. Theeffect of different surfactantsonthe structureand function of OmpTwas studied usingadetergent, DDM, andan amphipol, A8-35. Thechemical labelling technique hydroxyl radical footprinting (FPOP) wasused to oxidisethe solventaccessibleamino acid side-chains of OmpT.The protein wasthenproteolysed with trypsin and theresulting peptide fragmentsweresubjectedtoLC- MS/MSfor identification of theindividual modification sites(red structures). Thedatashowedthat whilst thedetergent DDMprotects only thetrans-membrane region of OmpT (blue circle),the amphipol A8-35protects boththe trans-membrane andthe extra-membrane regions(redoval).

www.astbury.leeds.ac.uk 2 main Publications Saunders J.C., YoungL.M., Mahood R.A.,Jackson M.P.,Revill C.H.,FosterR.J., SmithD.A., Ashcroft A.E., BrockwellD.J.&RadfordS.E.(2016)Anin vivo platform foridentifyinginhibitors of protein aggregation. Nat. Chem. Biol. 12:94-101.

Scarff C.A.,AshcroftA.E.&RadfordS.E.(2016)Characterizationofamyloid oligomersby electrospray ionization-ion mobilityspectrometry-mass spectrometry (ESI-IMS-MS). Methods Mol. Biol. 1345:115-132.

YoungL.M., Saunders J.C.,MahoodR.A., Revill C.H.,FosterR.J., Ashcroft A.E. &Radford S.E. (2016) ESI-IMS-MS:Amethod forrapid analysis of protein aggregationand its inhibition by smallmolecules. Methods 95:62-69.

SchiffrinB., CalabreseA.N., Devine P.W.A.,Harris S.A., Ashcroft A.E.,BrockwellD.J.& RadfordS.E.(2016)Skp is amultivalent chaperoneofouter-membraneproteins. Nat. Struct. Mol. Biol. 23:786-793.

IadanzaM.G.,Higgins A.J.,SchiffrinB., CalabreseA.N., BrockwellD.J.,Ashcroft A.E., RadfordS.E.&Ranson N.A. (2016) Lateralopening in theintact E-barrelassembly machinery captured by cryo-EM. Nat. Commun. 7:12865.

Dobson C.L., Devine P.W.A.,PhillipsJ.J., Higazi D.R.,Lloyd C.,Popovic B.,ArnoldJ., Buchanan A.,Lewis A.,GoodmanJ.,etal. (2016) Engineeringthe surface properties of a humanmonoclonal antibody preventsself-associationand rapidclearance in vivo. SciRep 6:38644.

Funding Ourresearchisfundedbythe BBSRC, theEPSRC,the WellcomeTrust,WatersUKLtd., LGC, AZ,UCB, &Medimmune. We thankthe BMSS forstudenttravelgrants.

Collaborators University of Leeds: D. Brockwell, R. Foster,S.Harris,P.Henderson,P.Kay,S.Radford,D. Rowlands,P.Stockley, N. Stonehouse, A. Wilson.

External: M. Quaglia (LGC,UK),M.Morris &K.Giles(Waters UK Ltd.), S. Macedo-Ribeiro (IBMC,Portugal), D. Raleigh(Stonybrook, NY,USA), J-L.Popot &M.Zoonens (CNRS Paris, France)and E. Duerling(Konstanz,Germany).

3 main Host- interactions of Risk Group 4 viruses

Rebecca Surtees, Amelia Shaw, JamelMankouri, Thomas Edwards andJohnBarr

Introduction Identification of interactionsbetween host andvirus components is importantasitholds the potentialtodevelopnovel diseaseprevention strategies that focusoneitherhostorviral targets. Therapeuticstrategiesthattargethostcellcomponents possibly reduce thelikelihood of resistance developing duetothe relativelyslowrate of cellular DNAgeneticchange comparedtothe more rapid time frameofvirus evolution. This is especially pertinentinthe case of RNA virusessuchasCrimean-Congo hemorrhagicfever virus(CCHFV),which have high mutationrates duetotheir error prone RNA polymerases. CCHFV is classifiedwithinthe Bunyaviridae family of tri-segmentednegativesense RNA viruses, whichcomprises five genera namely Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus and Tospovirus.The Nairovirus genuscontainsseveral serogroups,one of whichisthe Crimean-Congo hemorrhagicfevervirus (CCHFV) serogroupwithsolemembers CCHFV andthe genetically- distinct Hazara virus(HAZV)thatare formally groupedunder thesamespecies name of CCHFV.CCHFVisaRiskGroup 4human pathogen, responsiblefor adevastatingdisease forwhich preventativeortherapeutic measures do notexist.Transmission of CCHFV to humans is oftenbythe bite of infected ixodid ticksofthe Hyalomma genus, andthe human case-fatalityratecan exceed60%.Inrecent years, incidence of CCHFV-mediated disease hasbeennewly-reported in manyMediterraneancountries, likelyasaconsequenceofthe increasingly broad habitatand populationsizeofits tick vector,possiblyinresponse to climate change. CCHFV is nowrecognized as apotential threat to humanhealthinthe densely populated regionsofNorthernEurope, with recent fatalcasesinNorthern Spain causing concern. In contrast,HAZVhas notbeenassociated with serious humandisease,and is classified as aRiskGroup 2pathogen. HAZVisausefulsurrogate that canbeusedtostudy themolecular, cellularand disease biologyofthe highly pathogenicCCHFV,aswellasother nairovirusesresponsiblefor serious humanand animal diseases.

Results In ordertobetterunderstandthe interactionbetweenthe nairovirus Nproteinand thehost cell, we performedamass spectrometry-based analysis of its co-precipitating cellularproteins. Independentvalidationbyimmunological meanswithinfectiousHAZVand CCHFVrevealed that cellular chaperoneswere abundantinteractors, particularly thoseofthe HSP70familyand itsdnaJcofactors.Weshowedthese interactionswere maintainedwithinbothintracellularand extracellularvirions,suggestingthe interactions were required at multiple stages within the viruslifecycle.AblationofHSP70activityusingcharacterized small molecule inhibitors resulted in a1000-fold reductionofinfectiousHAZVproduction, with no evidence of virus escape.ThissuggestsHSP70family members provideanimportantrolewithinthe nairovirus replicationcycle,and that inhibitionofHSP70functionmaybe an important therapeutic

Figure 1: indirectconfocalimmunofluorescenceanalysisofSW13cells transiently expressing nativeCCHFV- Nprotein.Cells were co-stained using antibodiesspecificfor CCHFV N(green)and anti-HSP70 (red) and DAPI (blue).

www.astbury.leeds.ac.uk 4 main strategy forcurrently untreatable viraldiseases. We postulate that negative stranded RNA virusesare highly susceptible to errors in proteinfoldingthatmay arisedue to thehigherror rate of their polymerase.Such virusespossess RNP complexes built from multiple copiesof Nprotein in associationwiththe RNA genome,aswellasanassociatedpolymerase. The nairovirus Lsegmentisover12,000nucleotides long,and is likely encapsidatedbyover1000 Nproteinmonomersthatenwrapthe long Lsegment RNA. It is feasible that thenairoviruses havedeveloped astrategy involvingcellularchaperonestoprotectthissignificantmetabolic investmentinthe face of frequentpolymerase error.

Publications SurteesR., Dowall S.D., Shaw A.,ArmstrongS., Hewson R.,Carroll M.W.,MankouriJ., Edwards T.A.,Hiscox J.A. &BarrJ.N.(2016)Heatshock protein70family members interact withCrimean-Congo hemorrhagicfevervirus andHazara virusnucleocapsidproteinsand performafunctional role in thenairovirus replicationcycle. J. Virol. 90:9305-9316.

Funding This work wasfundedbyaPublic Health England/BBSRC CASE studentship.

Collaborators External: S. Dowall,R.Hewson,M.Carroll (Public HealthEngland)and S. Armstrong,J. Hiscox (University of Liverpool).

5 main Themany(interacting) facesofAurora-A kinase

Selena Burgess, Mark Richardsand Richard Bayliss

Introduction Aurora-Akinaseisbestknown forits role in theregulationofmitosis,specifically through the coordinationofprotein-proteininteractions that govern thetimingand robustnessofthe assemblingmitotic spindle. Thecatalytic activity of Aurora-A is stimulateduponinteraction withthe -associatedprotein TPX2,which promotes kinase autophosphorylation CrystalstructuresofAurora-A show howthisactivationprocessconvertsthe kinase froman inactive to an activeconformation.Indeed, unlikemanykinases that are regulated by phosphorylation, formation of afully-activekinaserequiresbothphosphorylationand binding of aprotein partner such as TPX2.Thisisbecause theactivationloop of Aurora-A is remarkably dynamic,and both phosphorylationand TPX2 bindingare required to lock it into a conformationthatiscompatiblewiththe bindingofsubstrates. More recently,Aurora-A wasfound to moonlightasaregulatorofMyc oncoproteins, afunction that is apparently independentofits kinaseactivity. Mycproteins are transcriptionfactors that markedly altergeneexpression through both activationand repression.The threehuman Myc proteins, c-Myc, N-Mycand L-Myc,haveregions of sequence homology that mediate interactions with critical partner proteins. Mycproteins areturnedoverrapidly,withashort half-lifeof~20 minutesinnon-transformed cells. Aurora-A stabilises N-Mycinneuroblastoma andc-Myc in livercancer, most likely by interfering with thedegradation of Mycthrough the ubiquitin-proteasomepathway.

Results Mycproteins areintrinsically disorderedand,consequently, thereare very fewpublished structural studiesonthem. We determined thecrystal structureofthe catalytic domain of Aurora-A boundtoN-Myc to 1.7 Åresolution(Figure 1).Residues61-89 of N-Mycare ordered, forminganextendedregionofcontacts that spanacross thesurface of Aurora-A. Basedon thestructure,wepropose that Aurora-Amustalterthe wayinwhich N-Mycisrecognised by theubiquitinationmachinery.

Figure 1: crystal structureofthe complex between Aurora-A (aa122-403) and N-Myc(aa61-89).N-Myc protein(red) interactswith theregionof Aurora-A (teal) that changesconformation between amore dynamic, inactive stateand alessdynamic,activestate.Here, Aurora-A is in an active conformation,the binding site forN-Myc is disruptedbyligandsthatstabilize an inactive conformation. Figure is basedonPDB entry5G1X.

TheAurora-Ainhibitoralistertibiscurrentlyunder investigationinclinical trialstotreat patients withneuroblastoma.The bindingofalisterib promotes theinactiveconformationofAurora-A, whichdestabilises theinteraction with N-Myc. Thestructure of theAurora-A/N-Myc complex explains whythe interaction is sensitive to Alisertib.Moreover, thestructure providesa template forstructure-guideddesignofsmall molecules that disruptthe interaction. This is an alternative,attractivestrategy forthe therapeutictargeting of Mycbecause alisertib is avery potentinhibitorofAurora-Akinaseactivity, butisnot particularly effectiveatdisruptingthe interactionwithMyc.

www.astbury.leeds.ac.uk 6 main Thestructural mechanism by whichTPX2activates Aurora-A hasbeenstudied formorethan tenyears.However, we have only recently madeprogress in using this information in the developmentofAurora-A inhibitors. Throughphage displayscreening of alibrary of single domain antibody (sdAb) scaffolds basedonasharkIgNARV, we identified vNAR-D01,asdAb that inhibitsAurora-Athrough an allostericmechanismthatisantagonistictothatofTPX2 (Figure2). Thecrystal structure of theAurora-A/vNAR-D01complexshows that thesdAb stabilises an inactive conformationofAurora-Ainwhich theLys-Glu salt-bridge,critical for activity,isbroken. We arenow exploring thedevelopmentofallosterickinaseinhibitorsbased on themechanismofaction of vNAR-D01. TPX2,likeMyc,isanintrinsically disordered proteinthatbecomes structured upon bindingto Aurora-A.Wegenerated asynthetic versionofTPX2thatincorporated ahydrocarbon-stapled helix.Stapled TPX2 retainedthe ability to activate Aurora-A,but boundtothe kinase with 10- fold enhancedaffinity.Thisstrategy could be used to developpeptideinhibitors of thevarious protein-proteininteractionsofAurora-A.

(a)(b)

Figure 2: Aurora-A canbepositivelyand negatively regulated throughinteractionsatthe same pocket. (a) Crystal structureofAurora-A(grey)incomplexwithTPX2(green).TPX2inserts apair of Tyrsidechainsintoapocket on thesurface of Aurora-A. This stabilisesthe active conformation, as indicatedbythe formation of aLys-Glu salt-bridge (blue-red spheres). (b) Crystal structureofAurora-Aincomplex with asynthetic sdAb,vNAR-D01 (yellow). ThesdAbinserts aTrp side chainintothe pocket on thesurface of Aurora-A. This stabilises a conformation in whichthe Lys-Glusalt-bridge is broken.

Publications RichardsM.W., BurgessS.G., Poon E.,Carstensen A.,Eilers M.,Chesler L.,&BaylissR. (2016) Structuralbasis of N-Mycbinding by Aurora-Aand itsdestabilizationbykinase inhibitors. Proc. Natl. Acad.Sci.USA. 113:13726-13731.

BurgessS.G.,OleksyA., CavazzaT., RichardsM.W., Vernos I.,Matthews D.,BaylissR. (2016) Allosteric inhibitionofAurora-AkinasebyasyntheticvNAR domain. Open Biol. 6: 160089.

Rennie Y.K.,McIntyreP.J., Akindele T., BaylissR., JamiesonA.G.(2016)ATPX2 Proteomimetic HasEnhanced Affinity forAurora-A DuetoHydrocarbon StaplingofaHelix. ACSChem. Biol. 11:3383-3390.

Funding This work wasfunded by Cancer Research UK, ERC, MRCand MRC-T.

Collaborators External: M. Eilers (Würzburg),L.Chesler (ICR, London), I. Vernos (CRG,Barcelona),D. Matthews (MRC-T),A.Jamieson(Glasgow),P.McIntyre(Leicester).

7 main Bio-inspired model membranesand light-harvesting nanomaterials

SanobarKhan, Mengqiu Li,StephenMuench,LarsJeukenand PaulBeales

Introduction Hybrid materialsaim to synergistically combinecomponents in ways that compensate fortheir individual weaknesses while maintainingand combiningtheir advantageous properties. Our aimistomakevesicle compartments with enhancedfunctionality andstabilityfor in vitro formulationand exploitationinbiotechnologies. Lipidvesicles,the most native-like reconstitutionsystem, have inherent biocompatibilityand biofunctionalityyet lack long term stabilityand versatilityinawiderange of environments.Conversely, polymersomes, vesicles that self-assemble fromamphiphilic blockcopolymers, whilelackingbiocompatibilityhave vastly superiormechanicaltoughnessand durability. Therefore hybrid vesicles composedof lipidsand blockcopolymersmight synergistically combine biocompatibilityand durabilityinto asinglematerial(Figure 1). This work develops andcharacteriseshybrid vesicleformulations of theredox-driven proton pump cytochrome bo3 ubiquinol oxidase(cyt bo3)asamodel system.Successfulenhancement of in vitromembrane proteinsystems will facilitate their applicationinwide-rangingapplications, includingbiosensors,pharmaceuticalassays, environmentalremediation andartificial cellsand organelles.

Figure 1: theredox-drivenprotonpump, cyt bo3, is reconstituted intohybridvesiclescomposedofa blend of lipidsand block copolymers.Our hypothesisisthatthesehybrids will synergistically combine thebiocompatibility and tough durability of theunitary materials. Thefunctionalactivityof cyt bo3 is assayedbyspectroscopically monitoring theoxidation of ubiquinol.

Results We developedanin vitro reconstitutionalprotocol forcyt bo3 inhybrid vesicles. Thelipid used in this studyfor POPC andthe polymerwas apolybutadiene-block-polyethyleneoxide(PBd22- b-PEO14). Initialmicellisationoflipidsand blockcopolymers usingdetergentsfollowedby detergentremoval in thepresenceofthe membrane proteinprovedunsuccessfuldue to the slow,viscousdynamicsofthe blockcopolymers frustrating themicelle to vesicletransition. Success wasfound in starting frompreformed hybrid vesicles that were destabilised with detergemttofacilitate membraneprotein insertion before removal of detergent by biobeads. Proteo-hybrid vesicles were createdacross thefullcompositionalparameter space from100% lipidto100%copolymer in 25%degrees of increment.Initial activity assays were conducted on theday thesamples were made.Proteo-liposomes showedthe greatest initialactivity (biofunctionality),while proteo-polymersomes were inactive.Significantly, therewas less than 20%decreaseinactivity betweenpurelipid systems and 50%copolymer vesicles. However formulations containing>50%copolymer saw arapid drop in initialactivity. Theadvantage of thehybrid systemsbecameevident when lookingatthe longer term functional activity of these proteo-vesicles over aperiodof6weeks. Thedecay rate of theinitial activityofthese vesicles significantlydecreased withincreasingpolymer content. Thebestcombinationofhighinitial activity andlongtermfunctionaldurabilitywas seen in the50% copolymer formulations.

However, an importantdetail in thecharacterisation of thesevesicles wasthatdynamic light scatteringsuggested thepresenceofapopulationof~20nm diametermicellescoexistingwith thehybrid vesicles in the50% and75% copolymersamples. Furtherinvestigationbycryo-

www.astbury.leeds.ac.uk 8 main TEMrevealedthatthere were in fact worm-likemicellesmulti-micronsinlength; theDLS appeared to be probingrelaxationtimes of internal modesinthese worm-likemicellesthat were consistentwiththe diffusion of 20 nm diameterBrownianspheres.

Hybrid vesicles were purified fromthese mixedsamplesbysizeexclusion chromatography on aSephadexG-50column, Analysisofeluted fractions proved theseparation of hybrid vesicle fractionsand that thefunctionalactivity of thesesamples wascontained within thesevesicles. Long term activity studieswere then repeated on purified 50%and 75% hybrid vesicles (Figure 2).75% copolymer hybrids hadsimilar functional decay ratesbefore or afterpurification. Howeverpurified50% copolymerhybridsappeartobemorestablethanthe initialmixture. Thesepurified50% hybridshaveaninitial activitydrop of ~25% in week one, likelydue to a less stable fractionforming in theinitial reconstitution;the activity then stabilises withamuch slower decay rate with 40%activityretained6months afterinitial reconstitution.This4-6 month half-lifecompareswitha1-2 week half-lifeinthe current gold standard of proteo- liposomes. Stored hybrid vesiclesamples willallow us to continue to monitor functional activity of thesesamples over a12-18 monthperiod.

Figure 2: longtermfunctional activity of cyt bo3 in proteo-hybridvesicles. Purified 50% (blue) and75% (magenta) hybrid vesicles retain significantactivity (~40% initial activity) after 6 months. In comparison, proteo-liposomes (grey dashed) haveafunctional half-life of 1-2weeksand areinactiveafter 3-4weeks.

We have shown that hybrid vesicles can increase thefunctionalhalf-life of amembrane protein by in excess of an orderofmagnitude.Thishighlysignificant enhancementinmembrane proteinfunctional durability hasthe potentialtorevolutionise their use in nascent biotechnologies. In particular, applications that were previously notviabledue to poor shelf- lifeorlongevity of functional efficacy may nowbecome feasible.

Publications Khan S.,LiM.Q., Muench S.P., JeukenL.J.C.&BealesP.A.(2016)Durableproteo-hybrid vesicles forthe extended functional lifetime of membraneproteinsinbionanotechnology. Chem.Commun. 52:11020-11023.

Funding This work wasfundedbythe EuropeanUnion MarieCurie Actionsand ERC.

9 main Protein engineering of aldolases

ClaireWindle,RobertSmith,AlexMoloney,NaimStiti,AdamNelson andAlanBerry

Introduction Aldolasesare attractivecandidatesfor useinbiocatalysis. They catalyse thealdol reaction and form newcarbon-carbonbonds with high specificity,highyieldsand undermild conditions. We areinterestedinre-engineeringthe substrate andstereochemical specificityofthese enzymes, using both traditional proteinengineeringtechniques andmethodswhich utilisenon- canonical amino acids.

Athreonine aldolase as atoolfor stereoselectivesynthesis 7KUHRQLQHDOGRODVHV KDYH HPHUJHG DV LPSRUWDQW WRROVLQWKH V\QWKHVLVRIȕ-hydroxy-Į-amino acids. Thesecompounds areoftenfound in natural productssuch as antibiotics and immunosuppressants. Recently,wehavedeveloped ahighthroughputscreentotarget librariesofE.coli threonine aldolase (ItaE) mutants createdusing CASTing(combinatorial activesitesaturationtesting). This approach hasthusfar been successfulinidentifying enzymeswithswitchedspecificities andenhancedselectivityfor thecleavagereactionof phenylserine (PS);althoughstill in theearly stages of screening(Figure 1).Ahigh resolution crystalstructure of thewild type enzyme has been solvedtohelpwithidentificationof importantactivesite residues.

Figure 1: steady-state kinetic parameters of thewild-type andmutantenzymes forthe cleavage reaction of 2S3R and 2S3S phenylserine.

Modifying substratespecificity usingnon-canonical aminoacids Until recently protein engineeringmethods were restrictedtousingthe 20 proteogenicamino acids to alterenzymeactivities. Ourrecentwork hasfocused on using non-canonical amino acids (ncAAs)toalter thesubstratespecificity of thealdolase N-acetylneuraminic acidlyase (NAL). ncAAs areincorporated at avarietyofpositions throughoutthe enzyme,using a method whichworksvia adehydroalanine intermediate andasubsequentadditionofathiol compound to form novelsidechains. ThesencAAcontainingenzymes have then been screenedfor alteredactivity.

When a2,3-dihydroxypropylcysteinewas positioned at theresidue190,the activityofthe enzyme forthe reaction betweenpyruvate anderythroseincreased abovethatofthe wild-type enzyme forthisreaction. This increase in activity could notbereplicated by using anyofthe canonical 20 aminoacids at this position,and using in silico modellingitwas discovered that thenon-canonical residueforms auniquehydrogenbondingnetwork that would notbe possibleusing canonical residues (Figure2).

We are expanding thesemethods to newenzymes, such as trans-O- hydroxybenzylidenepyruvate hydratase-aldolase.Weaim to generate highly efficientenzymes that catalyze fundamentallydifferentreactions to the native enzyme and/orwhich exhibit

www.astbury.leeds.ac.uk 10 main

Figure 2: leftshows theresult of in silico modelling forthe architecture of theactivesitewithsubstrate bound. Right shows theresults of thesamemodelling forthe ncAA containingenzyme, with thehydrogen bonding network highlighted.

modified substrate specificities. Combinationsofsuchenzymes mightbeexploited in one-pot, multisteptransformations, analogoustometabolic pathways that yieldhighvalue fine chemicalswhich arecovetedfor theirbiological activities.

With thesecomputationaland biochemicalmethods at hand, coupledwiththe enormous variety of side chainsand chemistries possiblewith ncAAs,the wayisnow open to engineer enzymeswithcatalytic functions that are notfound in Nature.

Publications Howard J.K.,MullerM., BerryA.&Nelson A. (2016) An enantio- anddiastereoselective chemoenzymatic synthesisofD-fluoro E-hydroxycarboxylic esters. Angew. Chem.-Int.Edit. 55:6766-6769.

Funding Ourwork is funded byBBSRC, GlaxoSmithKlineand Dr Reddy’s-Chirotech.

Collaborators External: J. Adams(GSK) andM.Bycroft (DrReddy’s-Chirotech).

11 main Engineering novel natural products and binding motifs

Ieva Drulyte, EmilyTurri,AdamNelson andAlanBerry

Introduction Natural productsbelongtoanextensive family of diverse organicmolecules: in excess of 200,000 discoveredand extractedfromvarioussources. Of particular interest arethe polyketide, non-ribosomalpeptideand isoprenoid classes, contributingtothe pharmaceutical, cosmetic andbiofuel industries.These classes of naturalproducts aresynthesised by polyketidesynthases, non-ribosomal peptidesynthases andterpene synthases, respectively; andour interest liesinunderstanding thestructure-function relationship of theseenzymes; facilitatingour engineeringeffortstosynthesise existing andnovel naturalproducts.

Structural studiesinindanomycin biosynthesis Polyketides represent abroad class of naturalproductswhich oftenpossess important biological andpharmacological activities. In nature,polyketidesare producedbymulti-enzyme proteins knownaspolyketidesynthases(PKS). Indanomycin, an antibioticactiveagainst Gram-positive bacteria,isproduced by ahybrid nonribosomalpeptide synthetase-polyketide synthase (NRPS/PKS) from Streptomycesantibioticus NRRL 81673.The aimofthisproject is to structurally characterisethe indanomycinPKS to gain abetterunderstanding of the interfacesbetween thefunctionaldomains, theassemblyofthe full complex andthe chemistry involved in thegeneration of mature polyketide(Figure 1).

Figure 1: indanomycin PKS with predictedcarrier- bound intermediates. PreliminaryEMand NMR dataofIdmO andIdmH, respectively,are also shown.

We have takenaninterestindetermining thestructure of IdmO,the fourth subunit of indanomycinPKS. IdmO hasbeen successfullyproducedand visualized by negativestain electron microscopy. Alow-resolutionmodel of IdmO wasproduced, whichsuggested a dimeric architecture.Going forward, alarge cryoEM datasetwillbecollected andprocessed to produce ahighresolution 3D structureofIdmO. We also studyIdmH,aputativepost-PKS cyclase enzyme thoughttocatalyze theindaneringformationvia aDiels-Alder [4+2] cycloadditionreaction. IdmH wascloned, heterologouslyexpressed andpurified. Biochemical analysis suggested IdmH also acts as adimer.3Dnuclearmagneticresonance spectra were collected whichallowed theassignmentofapproximately 65%ofbackbone resonances. Thesedatawill help to probebinding of IdmH to potentialsubstratesand furthercharacterize this enzyme.

www.astbury.leeds.ac.uk 12 main Engineering bisabolenesynthase fornovel activities Bisaboleneisasesquiterpenefound to have properties important forabiofuelinhydrogenatedform(bisabolane). Bisabolenesynthase producesbisabolenefromfarnesyl pyrophosphate bisaboylcation. Initialworkhas been focused on redesigningthe enzymeactivesite(Figure 2) to produce novelactivities andnovel terpenes. So far75enzymemutants have been designed andproducedusing site-directed mutagenesisand screened fornovel activities. Screening is carriedout using gaschromatography,massspectrometryand anynovel terpenes will be characterisedusing 2D NMR. The malachitegreen assayisbeingadapted fromthe work of Vardakoutoscreenthe mutants forpyrophosphate cleavage. This assayutilises an inorganic pyrophosphatase, converting Figure 2: crystalstructureof theactivesiteofAbiesgrandis pyrophosphate into twoinorganic ions,which bisabolene synthase (3SAE) consequently form themalachitegreen complex (Figure3). with farnesyl thio- Pyrophosphate cleavage is thefirst step in thereaction pyrophosphate(cyan)and mechanism, thereforethisassay can be usedtotestbothactive catalytically important andinactivemutants. Testingthe inactivemutants can tell us magnesiumions(green) bound. whetherthe cleavage of pyrophosphate is theinhibited step inactivatingthe enzyme. Fromthese 75mutants potentialcatalytically important residueshave been identified alongwithinactivemutants andmutationsincreasing thenativeproduct profile up to doublethe wild-type production level.

Figure 3: schematic diagram showing theadapted malachitegreen assay to screenbisabolenesynthase mutantsfor theproductionofpyrophosphate. Pyrophosphateiscleaved and released from thereactionwith bisabolene synthase and farnesyl pyrophosphate. Thepyrophosphateisconverted into twoinorganic phosphateionswhichreact with malachitegreen andmolybdatetoformthe malachitegreen complex.

Funding Ourwork is funded by BBSRCand TheWellcomeTrust.

13 main Economical and scalable synthesisof6-amino-2-cyanobenzothiazole

JacobHauser, Hester Beard, Stuart Warriner andRobinBon

Introduction 2-Cyanobenzothiazoles (CBTs) areusefulbuilding blocks forbothluciferin derivatives for bioluminescent imaging(BLI) andhandles forfast(k~10 M-1s-1)biorthogonalligations (Figure 1).Aparticularly versatileCBT is 6-amino-2-cyanobenzothiazole (ACBT, 9), whichhas an aminehandle forstraight-forwardderivatisation.PreviouslyreportedroutestoACBT arelow- yielding,difficulttoscaleup, and/or use costly andhighlytoxic (combinationsof) reagents. We developedasafe,economical andscalable synthesis of ACBT.

Figure 1: functionalised CBTs 1 canbeusedfor thesynthesisofluciferins 3 andfor bio-orthogonal ligations such as the site-specificlabelling/immobilisationofproteins 4.

Results Aftervariation of reagents, catalyst, solvent andreactiontemperature,wefound that the 2-cyanogroup of ACBT can be installedundermildconditionsthrough theDABCO-catalysed cyanationof2-chloro-6-nitro-benzothiazole 7 (Figure2). Anyunreacted cyanideinthe reaction mixturewas safely quenched by theadditionofanFeCl3 solution, andcalorimetricanalysis showed that therate andenergetic profile of thecyanationreactioncould be controlledbythe slow addition of adilute aqueousNaCNsolutiontothe reaction mixture, preventing potential thermal runaway upon scale-up. We also developedanimprovedprocedure forthe reduction of 2-cyano-6-nitro-benzothiazole 8 using iron powder.Our routeallowed thesafesynthesis of ACBT on multi-gram scaleand inhighpurity. In addition,the soleuse of filtrations and crystallisations forpurificationofall intermediatesand products,incombinationwiththe endothermicnatureofthe controlledcyanationprocedure,willenablestraight-forward further scale up if required.

Figure 2: optimised procedurefor ACBT synthesis.

Publications Hauser J.R.,Beard H.A.,BayanaM.E., JolleyK.E., Warriner S.L. &Bon R.S. (2016) Economicaland scalable synthesisof6-amino-2-cyanobenzothiazole. Beilstein J. Org. Chem. 12:2019-2025.

Funding We thankEPSRC forfunding (DTA studentshiptoHAB).

Collaborators University of Leeds:MaryBayanaand KatherineJolley(School of Chemistry).

www.astbury.leeds.ac.uk 14 main Structural studiesofauto-inhibition andmutationalactivation in fibroblast growth- factor receptors

BrendanFarrell, Gary Thompson, Arnout Kalverdaand Alex Breeze

Introduction Fibroblast growth factor receptors (FGFRs)are receptor tyrosine kinasesthatplaykey roles in embryogenesis, tissuedevelopmentand woundhealing,but arealsokey drivers of a number of humancancers.Weare usingNMR,alongside otherstructuraland biophysical techniques,tounderstanddisease-associated mutational activationofFGFRs at the molecularlevel,and howthisknowledge maybeused to developnew-generationdrugs against FGFR-mediated cancers.

Results Together with ourcollaboratorsatUCL (ProfMatildaKatan)and AstraZeneca,wehave explored thestructural andfunctionalconsequences of activatingmutationsinFGFR3 kinase domain in termsofthe effectsondrugresponses of clinical FGFR kinase inhibitors.We included in this comparison amutation, R669G, that lies close to,but outside, theactivation loop.

Figure 1: (a) positions of activatingcancer mutationsinFGFR3,displayed on FGFR1 crystalstructure. (b) amidechemicalshiftperturbations in FGFR3(R669G)relativetoFGFR3(WT); keysecondary structural and functional elementsare displayed above.

Themechanismwhereby R669Gactivates thekinasehas notpreviously been characterised, andour studiesusing X-raycrystallography andNMR have establishedthat, like thewell- knownK650E activationloop mutation,R669G stabilises an active-likeconformationofthe kinase activationloop,and inducescharacteristic amidechemical shift perturbations. Furthermore,our NMR-basedanalysisofthe conformationaland dynamiceffects of the R669G mutationdemonstratethatthere is ‘cross-talk’between thesitesofthe R669G mutation andthatofanother ‘hot-spot’ mutation, N540S, located30Åaway in theso-called ‘molecularbrake’region(Figure 1).Thisdemonstrates that spatially distantmutationsites are able to communicate structural anddynamic changes that affect theactivationstate of the kinase in similar ways viaallostericstructuralnetworks.

Publications Patani H., Bunney T.D.,ThiyagarajanN., Norman R.A., OggD., BreedJ., Ashford P., PottertonA., Edwards M.,Williams S.V.,etal. (2016) Landscapeofactivatingcancer mutationsinFGFR kinasesand their differential responses to inhibitors in clinical use. Oncotarget 7:24252-24268.

Funding This work wasfunded by CRUKand AstraZeneca UK.

15 main Structural biology of themicrotubulecytoskeleton

JoeCockburn

Howcellularcargo molecules recruitmolecular motors andregulate theiractivities Thecytoplasmisahighly crowdedenvironment containingtensofthousands of different proteinspecies, mRNA molecules, ribosomes, vesicles andorganelles. Cellularfunctionis critically dependent on thecorrect localisationofthese componentsinspace andtime.

Themovementofcellularcargoes over long distancesrequiresdedicated motor proteins (kinesinsand cytoplasmic )thatuse ATPhydrolysistopower movement alonga dynamic networkoftrackscalledmicrotubules. Howthese motors coupleATP hydrolysis to movement is nowfairlywellunderstood, andattention in thefield is nowturningtowardsthe questionsofhow cellularcargoes recruitmolecularmotors andregulate their motility. The combinedactionofall thekinesin anddynein motors inside your body is very powerful –ifall thekinesin motors in your cells were workingatfulltilt allthe time,theywould use up somewhereinthe region of 8000 kcal of energy perday!Molecularmotors must therefore be carefully regulated by theircargoes to ensure that they only consume energy then they are needed.

Themainfocusofour activityatpresentisonkinesin-1,which mediates thelong-range transport of diverse cellularcargoes (proteins, mRNPs,vesicles, organellesand viruses).We use structuralbiology,biophysical and cellbiology techniques to understand howkinesin-1 switchesitselfoff when notinuse,how cargo moleculesbindtokinesin,and howthis“switches on”-1.

Towards amolecular-levelunderstanding of theciliary transitionzone Cilia are theantennae of eukaryotic cells,sensing awidevarietyofenvironmentalsignals(e.g light, molecules, proteins, andfluid flow). Theciliumpossessesadistinctprotein andlipid composition relativetothe rest of thecell. This is maintainedbythe transition zone,alarge complexofover20proteinsatthe base of thecilium that controls theexchangeofmaterial betweenthe ciliumand therestofthe cell. Mutations in transition zone genesresultinarange of autosomalrecessiveinherited disorders,suchasnephronophthisis,Joubert Syndrome and Meckel-Gruber syndrome.Around1%ofthe populationare geneticcarriers forthese conditions.

Funded by aWellcomeTrust SeedAward, andincollaboration with Prof Colin Johnsonatthe Faculty of Medicine andHealth(University of Leeds), we will use acombination of structural andcellbiology approachestobegin to obtain aunified,molecular-levelunderstanding of the functionoftransitionzoneproteins,and howmutationsintransition zone genes cause diseases. This will aidinthe developmentofgenetherapiesagainst theseconditions.

Funding Fundingfromthe Wellcome Trust, theRoyal Society andstartupfunding from theUniversity of Leedsisgratefulyacknowledged.

Collaborators University of Leeds: C. Johnson, F. Sobot,M.Peckham.

www.astbury.leeds.ac.uk 16 main Thephysics of life in extremeenvironments

KasiaTych, MatthewBatchelor,ToniHoffmann,Michael Wilson,Megan Hughes,Samuel Lenton,Danielle Walsh, Natasha Rhys,James Towey, EmanuelePaci,David Brockwell and LornaDougan

Introduction We are developing single molecule manipulationtechniquesand neutrondiffractiontoexplore thephysics of living systems. Thesepowerfultechniquesare usedtostudy biomolecularself- assembly andthe structure anddynamicsofmoleculesinaqueous solutions, in both simple andcomplex systems.Weare particularly interested in thephysics of lifeunder extreme environmentalconditionsincluding high andlow temperatures,highsaltconcentrations and high and lowpH.

Results Extremophilesare organismswhich surviveand thriveinextreme environments.The proteins fromextremophilic single-celledorganisms arestructurally stable andfunctionallyactiveunder extreme physical andchemical conditions. Theseproteins provideexcellent modelsystems to determinethe role of non-covalentinteractions in defining proteinstabilityand dynamics as well as being attractive targets forthe developmentofrobustbiomaterials.Hyperthermophilic proteins have aprevalenceofsaltbridges,relative to theirmesophilic homologues,which are thoughttobeimportant forenhancedthermal stability. However, theimpactofsaltbridges on themechanical properties of proteins is farfromunderstood. We haveusedacombination of proteinengineering, biophysical characterisation, single molecule forcespectroscopy(SMFS) and moleculardynamics(MD)simulations to directly investigate theroleofsaltbridgesinthe mechanical stabilityoftwo cold shock proteins; BsCSPfromthe mesophilic Bacillus subtilis and TmCSP fromthe hyperthermophilic organism Thermotoga maritima (Figure1). We showthatagraftedionic clusterfromahyperthermophilic proteincan increase the mechanical softnessofamesophilic protein. We speculate that mechanical softness could provideamechanical recovery mechanismand that it maybeadesignfeatureapplicable to other proteins.

Figure 1: SMFS showsthat TmCSP is mechanically stronger yetits native statecan withstandgreater deformationbeforeunfoldingcomparedwith BsCSP. MD simulations were used to identify the locationand quantifythe populationsofsalt bridges, and revealedthat TmCSP contains alarger number of highly occupiedsalt bridges.Wetested the hypothesisthatsalt bridgesendow these mechanicalproperties by grafting an ioniccluster from TmCSP into BsCSP to design achargedtriple mutant (CTM).AsexpectedCTM is thermodynamically more stable andmechanically softerthan BsCSP.

As well as proteins,weare interested in understanding themolecularmechanismsbywhich thesolvent environmentcan stabiliseand protect moleculesand cells.Cryoprotectant moleculesare widely usedinbasic molecularresearchthrough to industrial andbiomedical applications. Themolecularmechanisms by whichcryoprotectants stabiliseand protect moleculesand cells,along with suppressing theformation of ice, are incompletelyunderstood. To gain greaterinsight, we completed experimentstodetermine thestructure of cryoprotectant solutions at lowtemperatures. Ourinvestigations combineneutron diffraction

17 main experimentswithisotopicsubstitutionand computationalmodelling to determinethe atomistic levelstructure of themixtures. We examinethe localstructureofthe systemincluding the water structure (Figure2).

Figure 2: we useacombinationofneutrondiffraction and computationalmodellingtoexamine thestructureofwater in glycerol-waterliquid mixtures at low temperaturesfrom285 to 238 K. We show that the mixtures are nanosegregated into regions of glycerol-rich andwater-rich clusters.Weexaminethe water structureand reveal that waterforms alow density waterstructure that is more tetrahedral than thestructureatroomtemperature. We postulate that nanosegregationallowswater to formsalow density structurethatisprotectedbyanextensiveand encapsulating glycerolinterface.

Publications Tych K.M.,BatchelorM., HoffmannT., Wilson M.C.,HughesM.L., Paci E.,Brockwell D.J. & Dougan L. (2016) Differentialeffects of hydrophobiccore packingresiduesfor thermodynamic andmechanical stability of ahyperthermophilicprotein. Langmuir 32:7392-7402.

Lenton S.,Walsh D.L.,RhysN.H., SoperA.K.&Dougan L. (2016) Structural evidence for solvent-stabilisationbyaspartic acidasamechanismfor halophilicproteinstabilityinhighsalt concentrations. Phys.Chem. Chem.Phys. 18:18054-18062.

Hughes M.L. &DouganL.(2016)The physicsofpullingpolyproteins: Areviewofsingle moleculeforce spectroscopyusing theAFM to studyprotein unfolding. Rep. Prog.Phys. 79:076601.

ToweyJ.J., SoperA.K.&Dougan L. (2016) Low-density water structure observed in a nanosegregated cryoprotectantsolutionatlow temperatures from 285to238 K. J. Phys. Chem.B120:4439-4448.

Tych K.M.,Batchelor M.,Hoffmann T.,Wilson M.C.,PaciE., BrockwellD.J.&Dougan L. (2016) Tuningprotein mechanicsthrough an ioniccluster graft fromanextremophilicprotein. Soft Matter 12:2688-2699.

Funding This work wasfundedbythe EuropeanResearch Council

Collaborators External: A. Soper(ISIS Facility,Rutherford Appleton Laboratories).

www.astbury.leeds.ac.uk 18 main Ferroptosisasanewcelldeathpathway involved in neurodegeneration

Andrew Tsatsanis,Bruce Wong and JamesDuce

Introduction Iron is required as acofactor in metabolicprocesses throughoutthe body andspecifically in tissues of high oxygen consumption, such as thecentralnervous system.The abilityofironto freelyreceiveand donate electrons is critical forneurotransmitterregulationaswellas oxidativephosphorylation, nitric oxidemetabolismand oxygen transport.Anironimbalance can lead to metabolicstress on theseprocesses. High levels of unboundironalso detrimentally catalyzethe productionoftoxic reactiveoxygenspecies. Increased cellular susceptibilitytooxidativestressassociated with iron accumulationleads to neurodegeneration withinpatients andanimalmodelsofdementiarelated disorders.

Iron Efflux through IronRetentionthrough Figure 1: schematicofAPP processing andiron APPnon-amyloidogenic APPamyloidogenicpathway . Non-amyloidogenic processing of APP pathway Tf Tf leading to increased efflux of iron through elevated APP Fe3+ sAPPs APP and FPN retention on thecellsurface.However FPN APP FPN amyloidogenicprocessingofAPP throughthe s -sec endocyticpathway leads to intracellular retention  -sec throughdecreaseFPN on the cellsurface. Inhibitionof 2+ Fe ARF6 HQGRF\WLFLQWHUQDOLVDWLRQ WKURXJK OLSLG UDIWVDQG ȕ- Fe2+ sAPP secretasetrafficking to theearly endosomehas also Ferroptosis been found to increaseAPP and FPN levelsonthe neuronal surface.

Cellular iron efflux is tightlycontrolledtomaintainironhomeostasis. We discovered anew endogenous regulatory mechanismfor AD-implicated amyloidprecursor protein(APP) in controlling neuronalironexport. APP facilitatesthe movementofironacrossthe plasma membrane by bindingtothe iron exporterferroportin(FPN) andpromotingits functional retention on thecellsurface.APP loss causes iron elevationand iron mediated celldeath. Restoringorreplacing neuronal iron export could be anovel mechanismofactionfor new therapeutics targetingoxidativestress in arange of neurodegenerative diseases. Ferroptosis is anon-apoptotic form of cell death that can be triggered by conditions that inhibitglutathione biosynthesis. This lethal process is definedbythe iron-dependent accumulation of lipid reactive oxygen species andanumberofsmall molecule inhibitors of ferroptosishavebeen identified(e.g. ferrostatin-1)which blockpathological celldeath in thebrain.

Results More recent developments in ourlineofresearch have resulted in anotherroute forAPP- dependent regulation of neuronal iron that is highly relevant to AD (Fig. 1). Secretase cleavage of APPisessentialfor APP maintenance on themembrane surface. Data currentlyinpreparation forsubmission indicatesthatalteringthe proteolytic processing of APP at thecellsurface causes consequentialchanges in neuronal iron homeostasis. Changing the expression,activityand cellulartrafficking of endogenous secretase activity to enhance the amyloidogenicpathway of APP processing (Fig.2A&B),oroverexpressing APP carrying familialADmutations(Fig. 2C), leadstointracellularironaccumulationvia cellsurface FPN changes.Withincreasedamyloidogenic processingofAPP beingamajorcontributor to AD, thesestudiesincrease ourunderstanding as to whyironaccumulationindementiapatients andtransgenicmodelscould facilitate susceptibility to reactiveoxygen species neurotoxicity. Persistentprocessing of APP by E-secretase to elevateneuronalironretentionleads to lipid peroxidation andferroptosisinhibition by Ferrostatin-1 is able to protectagainst celldeath inducedbyaltered APP processing (Fig.2D).Combined, this evidence provides an intriguing

19 main therapeutictargetfor dementia associated with abnormalproteolytic processing of APP.

A B Figure 2: APP proteolysisaltersneuronaliron homeostasis with ferroptosis inducedby amyloidogenicprocessing of APP beingrescued by chelationand ferrostatin-1. (A,B)Altering proteolyticcleavage of APP causes aresponse to intraneuronal iron levels (A)and ferroportin(FPN) cell surfacelocation(flow cytometry)(B  Į- CDSecretaseinhibition (TAPI-2) increases amyloidogenicprocessing of APP andiron UHWHQWLRQ ZKHUHDV E\ ȕ-secretase inhibition (bIV) inducesnon-amyloidogenicprocessing of APP, reduces iron and increasescell surfaceretention of FPN. (C)Similarto(A) amyloidogenic processing of APP caused by theADfamilialItalian mutation increased intraneuronal iron levelswhereas thelongevityIcelandic (Ice)mutationassociatedwith LPSDLUHG ȕ-VHFUHWDVHSURFHVVLQJUHWDLQHGLURQDWFRQWUROOHYHOVȕ-secretase inhibition(bIV)only reduced iron fromcelllines overexpressing wild-type APP or Ita-APP. (D)All iron inducedcell deathwas preventedby addition of deferiprone (DFP), whereas ferroptosis inhibition(ferrostatin-1;Fer-1) rescued cell toxicity only inducedthrough amyloidogenicprocessingofAPP (TAPI-1). ***, p<0.001; **,p<0.01; *, p<0.05 compared to control. ForCand D^^^,p<0.001; ^^, p<0.01;^,p<0.05comparedtoirontreated.

Outlook By continuingtosupport anovel candidatefunctionfor APP we nowbegin to explainthe diverse trophic andmorpho-regulatory activitiesofthe protein andelucidate thevulnerability thebodytoage-associated ironaccumulation. Modulatoryfactors that can increase theprocessing of APPthrough theamyloidogenic pathwaymay have additional affects on iron regulationand oxidativestress. We have identifiedthatferroptosis maybeaninstrumentalconsequence of chronic altered amyloidogenicprocessing of APP. Similar to arange of neurodegenerative models identified by collaborators, we have nowidentifiedferroptosis as akey player in diseaseprogression andthe use of feroptotic inhibitionasapromising futuretherapeutic target forthese diseases.

Publications PoujoisA., DevedjianJ.C.,MoreauC., DevosD., Chaine P.,Woimant F. &Duce J.A. (2016) Bioavailabletrace metalsinneurological diseases. Curr.Treat. Options Neurol. 18:46.

Gunn A.P.,WongB.X.,JohanssenT., Griffith J.C.,Masters C.L.,Bush A.I.,BarnhamK.J., Duce J.A. &ChernyR.A.(2016)Amyloid-peptideA3PE-42 induceslipid peroxidation, membrane permeabilization, andcalcium influx in neurons. J. Biol. Chem. 291:6134-6145.

Funding This work wassupportedinthe UK by aPhD StudentshipfromAlzhiemer’s Society anda Marie Curie IntegrationGrantfromthe EuropeanCommission. International support is also providedbythe NHMRCand AADRFProject GrantfromAustralia.

Collaborators External: A. Bush (University of Melbourne, Australia); D. Devosand Dr.J.-C. Devedjian (Universite de Lille, France); R. Evans(Brunel University, London);D.Tetard andF.Lewis (Northumbria University) andD.Smith (SheffieldHallamUniversity).

www.astbury.leeds.ac.uk 20 main Structure-based design of inhibitors of metallo E-lactamases: rescuingour current antibiotics

Ricky Cain and Colin Fishwick

Introduction The E-lactamantibiotics such as thepenicillinsand cephalosporins, remain themostimportant drugclass forthe treatment of bacterialinfections.However, their continueduse is jeopardized by theincreasing spread of resistance mechanisms,including that mediatedbyE-lactamases, which, cumulatively, canhydrolyseall classesofE-lactamantibiotics. Theserine-E- lactamases(SBLs), classes A, C,and D,likelyevolved from thepenicillin-binding protein (PBP) targets of E-lactamantibiotics. Inhibitors of theSBLsincludeclavulanicacid, sulbactam, andtazobactam, whichare active against class A E-lactamases, andthe recently introduced non-E-lactam E-lactamaseinhibitor avibactam, whichhas abroaderspectrumofSBL inhibition activity. Theseinhibitors have increased theefficacy of E-lactamantibiotics againstSBL- mediated resistance in bacteria,but they are inactive againstthe Zn(II)-dependent class B metallo-E-lactamases(MBLs), whichconstitute astructural andmechanistically distinctfamily of enzymes andexhibit considerableheterogeneity,evenamong themselves. TheMBLsare able to hydrolyseall classesofE-lactamexceptfor monobactams.The ability of theMBLsto hydrolyseSBL inhibitors, includingavibactam,isagrowingprobleminthe treatment of infections wherebothSBL-and MBL-mediated cephalosporin andcarbapenem resistance have been acquired.Todatethere arenoclinically approved MBLinhibitors. Ourworkaims to usestructure-guided moleculardesignand organicsynthesisinordertoidentifysmall molecule inhibitors of E-lactamasesthatinthe form of aco-therapy, have thepotential to render resistantbacteriaopentothe effectsofE-lactam antibiotics.

Results We appliedthe de novo moleculardesignprograme SPROUT to acrystal structure of NDM-1 (4RAM.pdb) followed by subsequent synthesis, to produce asmal libraryofcyclic boronate- based inhibitors. Usingafluorogenic assayfor MBLs, thesewerethenscreenedagainst a representativepanel of clinically relevantB1subfamily MBLs, includingIMP-1 (Imipenemase- 1), VIM-2(Verona-Integron-EncodedMBL-2), NDM-1(NewDelhi MBL-1), SPM-1(SãoPaulo MBL-1), andthe modelMBL,BcIIfrom Bacilluscereus (Table 1).

IC50 [µM] 1234 5 BcII 7.27 0.30 0.96 0.52 1.14 VIM-2 0.0510.003 0.0110.014 0.002 IMP-1 1.44 1.00 1.50 1.21 1.41 NDM-12.040.029 0.687 0.04 0.004 SPM-124.116.716.013.936.3 TEM-10.001 0.0030.002 0.00030.006 OXOXAA-1-10000.33.3355.1.1 00.83.83 2.26 12.7 Figure 1: X-raycrystal structures of 2bound to theVIM-2 (left) and OXa-10 (right).

Theresultsimply that cyclic boronates with an aromatic sidechain,positioned analogously to WKHȕȕ VLGHFKDLQVRIWKH SHQLFLOOLQVFHSKDORVSRULQV DUH SRWHQW LQKLELWRUV RI % 0%/V ,Q vitro inhibitionofMBLsbythe tested cyclic boronates yieldedthe following rank orderof

21 main potency: VIM-2 > NDM-1 > BcII > IMP-1 > SPM-1. Overall, these data identify 2 and 5 as highly potent inhibitors of VIM-2and NDM-1, themostwidely distributedmembers of the clinically important B1 subfamily. We then usedfluorogenicassays to measure thepotency of thecyclic boronates against clinically relevant Class Aand Class DSBLs, includingTEM-1 (Class A) andOXA-10(Class D).All of thecompounds tested were potent TEM-1inhibitors (IC50 ĺ Q0 DQG FRPSRXQGVZLWKVDWXUated sidechains(1and 3)manifestedIC50 values <1µM against OXA-10.

We were able to verify themodeofbinding of theseinhibitors to both classes of enzymes via X-raycrystallography (Figure1).

Figure 2: X-raycrystal structures of 2bound to theVIM-2 (left) andOXa-10 (right).

Since 2 wasapotent inhibitorofbothenzymeclasses in vitro,wenexttestedits activity against highly resistant Gram-negative bacterialcells (strains of E. coli and Klebsiella pneumoniae, both carryingthe NDM-1MBL together with multiple SBLsofdifferentclasses),alone andin combinationwiththe carbapenemmeropenem,Compound 2 alonedid notdisplay antibacterialactivityagainst anyofthe strainstested.However, forall strainscarryingthe MBL NDM-1, co-administrationwith 2 reducedthe minimalinhibitory concentration(MIC) of meropenem.Clear reductions in meropenemMIC were observed at 10 Pg/mL inhibitor, while increasingthe concentrationof2to 25 Pg/mL broughtthe meropenemMIC into the susceptible range(MIC<8Pg/mL). Strikingly,for theclinical strain K. pneumoniae IR16, compound 2,evenat10Pg/mL,was able to reduce themeropenemMIC from resistant(32 Pg/mL)tofully susceptiblH 0,&” Pg/mL). Neithercompounds 1,nor 2 showed F\WRWR[LFLW\LQKXPDQ +(. FHOOV ZKHQ DGPLQLVWHUHG DW FRQFHQWUDWLRQVXSWR ȝ0

Publications AdamsP.G.,Collins A.M.,Sahin T.,SubramanianV., UrbanV.S., Vairaprakash P., Tian Y., EvansD.G., Shreve A.P. &Montano G.A.(2015)Diblock copolymer micellesand supported filmswithnoncovalentlyincorporated chromophores: Amodular platform forefficientenergy transfer. Nano Lett. 15:2422-2428.

AdamsP.G., SwingleK.L., Paxton W.F.,Nogan J.J., StrombergL.R., FirestoneM.A., Mukundan H. &Montano G.A. (2015)Exploitinglipopolysaccharide-induced deformation of lipid bilayers to modifymembrane compositionand generate two-dimensional geometric membrane arraypatterns. Sci. Rep. 5:10331.

Funding This work wasfundedbythe MRC.

Collaborators External: C. Schofield(Oxford), J. Spencer(Bristol).

www.astbury.leeds.ac.uk 22 main

Newsmall moleculeinhibitors of T. gondiibc1:towards acurefor toxoplasmosis

Martin McPhillie, James Gordon,StephenMuench andColinFishwick

Introduction Toxoplasma gondii,the causativeagent of toxoplasmosis, is responsiblefor themostcommon parasiticinfection of humanbrain andeye,persistsacrosslifetimes, can progressively damage sight, andiscurrently incurable. T. gondii infections can cause systemicsymptoms, damage anddestroy tissuesespecially eyeand brain, andcause fatalities. Primary infections maybeasymptomatic,orcause fever, headache, malaise, lymphadenopathy,and rarely meningoencephalitis,myocarditis,orpericarditis. Retinochoroiditisand retinalscarsdevelop in up to 30%ofinfected persons, andepilepsymay occur. Furthermore,expression of the essentialmitochondrial electron transportcomplex cytochrome bc1,ismarkedlyincreased in infectiveforms of theparasite,suggestingcytochrome bc1 mightbeaviable drugtargetfor thedevelopment of agents to combat toxoplasmosis.

We wishedtouse astructure-guidedapproach to produce potent smallmolecule inhibitors of T. gondii bc1 that mayrepresent starting points fordevelopmentinto anti-infectivedrugleads.

Results We appliedarangeofstructure-guidedmethods to a homology model of T. gondiibc1complexderived from thecrystalstructureofbc1 from S. cerevisiae (1KB9.pdb, Figure 1),and used structure-based methodsfollowedbysynthesistoproduce aseriesof putative bc1 inhibitors.

Oneofthese inhibitors,MJM170, wasfound to be a potent inhibitor (IC50 =30nMofthe growth of T. gondii tachyzoites ex vivo (Figure2).

Furthermore,wefound that MJM170 washighly efficaciousagainst T. gondii tachyzoites in miceat25 mg/kgwithout toxicity for5days (Figure3). Figure 1: X-raycrystal structureofbc1 from S. cerevisiae (1KB9.pdb)

Figure 2: structureofMJM170 and T. gondii tachyzoiteinhibitory data

23 main

Figure 3: in vivo effectofMJM170. MJM170 25 mg/kgdaily reduces type 2parasites.

We also usedX-ray crystallography to establishthatMJM170binds into theQisiteofbc1 (Figure4).

Figure 4: crystalstructureofMJM170bound into theQisiteofbc1 (bovineenzyme)

Publications McPhillie, M.,Zhou, Y.,ElBissati,K., Dubey, J.,Lorenzi,H., Capper, M.,Lukens, A., Hickman,M., Muench,S., KumarVerma,S., et al.(2016)New paradigms forunderstanding andstepchanges in treating activeand chronic, persistentapicomplexan infections. Sci. Rep. 6:29179.

Funding This work wasfunded by theNIH.

Collaborators External: R. McLeod (Chicago).

www.astbury.leeds.ac.uk 24 main Understandinghow thefusionprotein of Herpes SimplexVirus-1,gB, drivesfusion betweenthe virus and thehostcell membranes

Lauren Brownand JuanFontana

Introduction HerpesSimplex Virus(HSV) is amodel system forthe Herpesvirusfamily, whichincludes humanviruses that cause life-long infections andavariety of diseases,includingskinlesions, encephalitis andcancers.Antiviralsthatreduce theseverity andfrequency of HSV symptoms exist. However, thesedrugs cannotcureinfection,and thereisnoHSV vaccine available. Akey step of viralinfection is entryinto thehostcell, aprocess that forenveloped viruseslike HSV involves fusion of viraland cellularmembranes, allowingthe viralgenometoaccess the interiorofthe cell. In HSV, this processismediatedbythe fusion proteingB, forwhich a structure in itspost-fusion conformationisavailable.While this structurehas significantly advanced ourunderstandingofthe fusion process of HSV,itisthe structureofpre-fusion gB that is essentialtocomplete ourknowledge of howgBundergoes thepre-topost-fusion transition. However, solving this structure hasbeenhamperedbythe fact that allofthe purified formsofgBare post-fusion.

Results We have set-upasystem that generatesvesicles displayingfull-length gB from HSV type 1 on their envelopeand analysed them by cryo-electrontomography(cryo-ET), whichallows direct 3D imagingofuniqueobjects,combinedwithsubtomogram averaging,which further improvesthe signal to noiseratio of repeated structures within atomogram,likefusion proteins withinavirion.Toelucidate therearrangement of gB during fusion, we have labelledgB’s domainsbyinserting fluorescent proteins(whichare readilyvisible in thesubtomogram averages) in specificpositions andwehaveco-expressed gB with neutralisingantibodies (whose size is comparabletothatofthe fluorescent proteins) forwhich thebindingsites are known. Theseexperiments have provided aworkinghypothesis forthe structure of pre-fusion gB andthe conformations rearrangements of gB during fusion.

Figure 1: Cryo-ET (A,B,D,E,G&H;scale bars50nm) &subtomogram averaging(C&F;scale bars, 5 nm)offull-lengthgBexpressedinvesicles.A-C, gB WT.D-F, gB tagged with cyan fluorescent proteinat residue100.G&H,gBco-expressedwiththe neutralising antibody SS55.

Funding This work wasfundedbythe University of Leedsand theRoyal Society.

Collaborators External: D. Atanasiu,W.-T. Saw, R. J. Eisenberg andG.Cohen (University of Pennsylvania).

25 main Developmentofanovel smallmolecule anticoagulantwithminimal bleeding risk

RogerTaylor, ColinFishwick, CharlotteRevill,EmmaHethershaw andRichard Foster

Introduction: Theunderlyingfocusofour research is smallmolecule drugdiscovery.Weare interested in thesynthesis anddevelopment of smallmoleculesastherapeuticsaswellasthe design of chemical probes to support validation of novelbiological targetsinareas of unmetmedical need.Apriority area of research is development of newapproachestothe generationofsmall molecule anti-coagulants with minimalbleeding risk.

Results: We have identified potent,novel smallmoleculeinhibitors of thecoagulationcascadewith exceptional in vivo efficacy.The inhibitors have been identified by anumberofparallel approaches incorporatingvirtual drug design,chemical synthesis andhigh-throughput screening of drug-likesmall molecule librariesand fragment screening.Presently,weare optimising theinhibitors fortargetpotency,specificity anddrug-like physicochemical properties using iterativeroundsofmedicinal chemistry developmentand screening usinga paneloforthogonal bioassays. Thecompoundshavebeenoptimised fordrug-likenessand demonstrate high aqueoussolubility, metabolic stability, plasmastability andlow levels of plasma protein bindingand cardiotoxicity,includinghERGinhbition.The compounds are >300x selectivefor 10 structurally (and functionally) related targetsand thecompounds demonstrate no toxicity on high concentration dosing in mice. Theaim of theon-going work is to further developthe leadstogenerate acandidate compoundwithsuitablepharmaceutical properties consistentwithanoptimised lead ready forprogressiontoout-licensing and eventuallyclinical trials. Asecond seriesofinhibitors identified by fragment-based screening (NMR andSPR)isundergoingoptimisation forpotency driven by structural guided design. Thesecond series of ligands bind in adifferent manner to theprimaryseries andoffera possibilitytogenerate inhibitors with acomplementary selectivityand property profile to the currentleads.

Publication Patent:15217111

Funding: Ourwork is fundedbythe Wellcome Trust, MRCand BHF.

Collaborators University of Leeds: R. Ariens andH.Philippou.

www.astbury.leeds.ac.uk 26 main Structuraland functional studiesofmembrane pyrophosphatases

CraigWilkinson, NitaShah, Steven Harborne andAdrian Goldman

Introduction Membranepyrophosphatases (M-PPases) occur in plants,protozoan parasitesand prokaryotes.Theyare associatedwithlow-energystress: overcomingsalineordrought conditionsinplantsorrapid changes in pH or osmotic pressureinprotozoanparasites. M- PPasescouplepyrophosphate hydrolysis to thepumping of sodiumionsand/orprotons across theinner membrane of prokaryotes or thevacuole/acidocalcisome membranes of plants/parasites. These proteinsare importantinthe lifecycle of several pathogenic species of parasites, including Plasmodium falciparum,which causes malaria,and in several species of pathogenic bacteria,such as Clostridiumbotulinum andspecies of Bacteroides that can cause severe opportunisticinfections.There arenoanalogues in mammals, so M-PPasesare an importantpotential drug target.Continuingfromour M-PPase structure,weare completing amodel of thecatalytic cycleusing additional structural andbiochemicaldataand areusing this in drug design.

Results We have solvedtwo newstructuresofthe M-PPase of thethermophilic bacteria: Thermotoga maritima (TmPPase) in differentcatalytic states2.Combining this data with previouslysolved structuresofthisand arelated protein from Vignaradiata (VrPPase)wehavegenerated a model of thecomplete catalytic cyclecoveringall of themajor catalytic states (Fig.1). Newstructural information confirmed that thereisa “downwards” of helix12upon substrate bindingthat severs an important ionpair at thebaseof theactivesite. This allows theactivesite residues to alter conformation, leading to thecomplete coordinationofa nucleophilic water by twoaspartate residues, setting up themechanismfor Figure 1: thecatalyticcycle modelproposed formembranepyrophosphatases hydrolysis.The new based on structural informationfromX-raycrystallography.Onlykey helicesand structure also shows residues areshownfor thesakeofclarity. abound Na+ at theion gate,however, from thestructuresalone it is notpossibletotellwhether ionpumping or hydrolysis occurs first during this high-energytransitionstate.Understanding this state would provideinformation on this final, importantstepofthe catalyticcycle.Totry tosolve this problem, electrometric measurementswere taken of VrPPase proteoliposomes using theNanionSURFE2RN12, whichcan measure differencesinchargeacrossamembrane.Experiments involvingthe substrate(pyrophosphate) (Fig.2)generated astrongand repeatable signal attributedto proton pumpingthatwas absentinphosphate-only,and emptyliposome controls(Fig.2A). Further experimentsusednon-hydrolysable inhibitors in place of pyrophosphate (Fig.2B) and producedanabove-background signal that could be dissipated by protonophores. This

27 main suggeststhatthe signal seen with theinhibitors is duetoasingle-turnover event uponinhibitor bindingand that ionpumping occurs aftersubstrate bindingand before substratehydrolysis.

FigureFig 2: electrometric meameasurementsofVrPPase proproteoliposomes using the Nanion SURFE2RN1. CurreCur ntsobtained following the additionadd of:(A) K4PPi,K4PPi + CCCCCCP and K2HPO4;(B) IDP, IDP +CCCPand K2HPO4.All cocommpounds were added in the aactctivivaatingbuffer at the1- seseccond time point.

We arecurrently studying otherclassesofM-PPasessuchasthe K+-independent H+-PPases andthe dual-pumping PPasesfrom Bacteroidesvulgatus,using cryoEM to solve these structures andthose of othermembraneproteins. Biochemical andfluorescence studies, alongsidemoleculardynamicssimulations,are beingusedtounderstandthe mechanisms involved in eachstage of thecatalytic cycle. Finally, we arestartingtodevelop potentiallead moleculesthatkillprotozoanparasitesincollaborationwithlabsinLeedsand Helsinki.

Publications Shah N.R.,VidilaserisK., XhaardH.&GoldmanA.(2016)Integral membrane pyrophosphatases: Anovel drug target forhuman pathogens? AIMSBiophys. 3:171-194.

Li K.M.,Wilkinson C., Kellosalo J.,TsaiJ.Y., Kajander T., JeukenL.J.C., SunY.J.&Goldman A. (2016) Membranepyrophosphatases from Thermotoga maritima and Vignaradiata suggest aconserved couplingmechanism. Nat. Commun. 7:13596.

Goldman,A., BoijeafGennis G.,Xhaard H.,MeriS.&Yli-KauhaluomaJ.(2016)Pyrofosfaatti lääketieteessä. Duodecim 132:1111–1117(In Finnish).

Ji Y.R., Postis V.L.G.,WangY.Y.,Bartlam M. &Goldman A. (2016) Transport mechanism of aglutamatetransporterhomologue Glt(Ph). Biochem. Soc. Trans. 44:898-904.

LeeS.C., KnowlesT.J.,Postis V.L.G., JamshadM., ParslowR.A., LinY.P., GoldmanA., SridharP., Overduin M.,MuenchS.P.,etal. (2016) Amethodfor detergent-free isolationof membrane proteins in theirlocallipid environment. Nat.Protoc. 11:1149-1162.

Parmar M.J.,Lousa C.D.,MuenchS.P., GoldmanA.&Postis V.L.G. (2016)Artificial membranes formembrane proteinpurification, functionalityand structurestudies. Biochem. Soc. Trans. 44:877-882.

Funding BBSRC(ALERT-13and BB/M021610/1), RoyalSociety (Wolfson award), Wellcome Trust (091322/2/10/2), EDICT(201924), AcademyofFinland (12522061and 256049),Sigrid Juseliusfoundationand Leeds110th Anniversary scholarship

Collaborators University of Leeds: L. Jeuken,R.Tuma,S.Harris, C. Fishwick; on otherprojects A. Morgan. External: J. Kellosalo,T.Kajander, G. BoijeafGennäs andJ.Yliakauhaluoma (Helsinki),Y.- J. Sun(Taiwan), D. Linke(Oslo)and R. Lahti(Turku).

www.astbury.leeds.ac.uk 28 main Causativeand curativeviruses in human cancer

AbigailBloy, MatthewBentham,ClaireScott,HannahBeaumont, Emma Brown, Elizabeth Appleton, Mohamed Madkour, Adel Samson andStephen Griffin

Introduction Virusinfectionsare associated with 20-30% of human cancer,withevidence of their further involvementarisingthroughthe genomics era.However, whilst certainwell-established oncogenic DNAvirusescause cancervia expressionofdominantoncogenes or integration of theviral genomewithinhostDNA,the mechanisms by whichsome other virusesdosoare less apparent,despite clearassociations with malignantchange.

Hepatitis Cvirus (HCV) infects over 150millionindividuals andisassociatedwithapprox.30% of primaryliver cancer,predominantlyhepatocellularcarcinoma (HCC).However, as an RNA virusthatreplicates within thecytoplasm,its direct oncogenicmechanismisunclear, and dogma attributes HCV-associatedHCC to aconsequence of chronicinflammation. Nevertheless, growingevidencesupports thatHCV directly drives transformationand that treatingboththe cancerand virusconcomitantly maybebeneficialfor patients.

Ourresearchinthisareacomprises twoparallelthemes:firstly,weare investigatingthe mechanisms by whichHCV drives oncogenic change. Secondly,bycontrast,weare investigatingthe use of benign virusesasanimmunotherapy targetingvirus-associated cancer –so-called“oncolytic”virus immunotherapy.

Results 1. Mechanisms of HCV-inducedcarcinogenesis. Difficulties in culturingHCV in the laboratory have preventedresearchers fromconductingclassical assayswhereby prolonged virusinfection of primarycells leadstoimmortalisationand transformation.Nevertheless, evidence from transgenic systems shows that HCVproteinscan transformcells.Itisoften assumedthatHCV inducesthe formation of acancer-initiatingcellfollowing infectionof terminally differentiated hepatocytes. However, themajority of cells within achronically inflamed liver areinfact beingconstantlyrepopulated throughthe proliferationofthe resident hepaticprogenitorcell(HPC) compartment.

Figure 1: HCV-induceddelay of hepatic differentiation.HCV infection of Huh7 cellsperturbsexperimental differentiation,leadingtoaltered morphology (phase), lowered expression of terminaldifferentiation markers (albumin) and increasednumbers of cellsincycle (Ki67).

We hypothesised that HCVmight infect HPC, perturbingtheir proliferationand differentiation programmesand so increasing thelikelihoodoftransformation. In agreementwiththisnotion, we have shown that primary humanHPC isolated fromhealthy liver are susceptible to HCV infection ex vivo,setting aprecedent forthese representinganalternativesourceofcancer initiatingcells.Moreover, HCV significantlydelays andperturbshepatic differentiationincell

29 main culture models,maintaining cells in cycledespitestrongdifferentiationstimuli (Figure1). Ongoing work implicatesHIPPO signallingasapathwayhijackedbythe virusinorder to achieve this effect.Weare currently exploringthe precise mechanisms at play duringthis phenomenon,includingassessing suchvirus-inducedeffectsuponprimary HPC.

2. Oncolytic virusimmunotherapyinvirus-associated livercancer. It is clearthathyper- activationofour immunesystems,or“immunotherapy”,representsapotent newway of improving cancertreatment.Thiscould be particularly important forvirus-associated tumours whereinflammatory responsesshouldconcomitantly target both theunderlyingoncogenic virusinfection,aswellasmediatingimmune lysisofcancerouscells.Ironically, onepotent meansofachieving this objectiveisthroughtherapeutic administrationofotherwisebenign “oncolytic”viruses (OV). Classically,OVwere thoughttoact principally viapreferential replicationwithinimmune-compromisedcancerouscells,leading to direct tumour lysis. However, it is nowincreasingly recognised that these insteadact primarily viaimmune stimulation.

We showed that clinical grade oncolytichuman Orthoreovirus (Type3,Dearing,“Reo”)elicited potent type 1interferonresponses using in vivo preclinical modelsofHCC.These responses simultaneouslyactivated naturalkillercells to directly lyse tumours,whilstsuppressing the replicationofHCV.Thistranslated to significantsurvival benefits,and wasalsoapplicable to other modelsofvirus-associated cancer (hepatitisBvirusHCC, Epstein Barr viruslymphoma). Theefficacy of uv-inactivated virusconfirmedthisprocesstobeprincipally immune-mediated ratherthancytopathic. We have since developedsynergistic regimens baseduponmodified ReocombinedwithtargetedHCC therapies, whichdramatically enhance survival in preclinical models, andweare investigatingthe innate immune activationassociated with beneficial outcomes.

Publications SamsonA., BenthamM.J., ScottK., NuovoG., BloyA., Appleton E.,Adair R.A.,DaveR., Peckham-CooperA., ToogoodG., Nagamori S.,Coffey M.,Vile R.,HarringtonK., SelbyP., Errington-Mais F., MelcherA.A.&Griffin S. (2016) Oncolytic virusesascombined anti-viral andanti-tumouragentsfor thetreatment of liver cancer. Gut doi: 10.1136/gutjnl-2016-312009.

Mankouri J.,WalterC., Stewart H., BenthamM., Park W.S.,Heo W.D., Fukuda M.,Griffin S. &HarrisM.(2016) Releaseofinfectioushepatitis cvirus fromhuh7cells occurs viaatrans- golginetwork-to-endosome pathwayindependentofvery-low-density lipoproteinsecretion. J. Virol. 90:7159-7170.

Funding This work hasbeenfundedbyCRUK, MRC, ERCand theUniversity of Leeds

Collaborators University of Leeds: A. Macdonald(SMCB), R. Foster (Chemistry), G. Cook (LICAP),G. Toogood(SJUH). External: A. Melcher(ICR),W.Barclay (Imperial),W.Fischer (Taipei).

www.astbury.leeds.ac.uk 30 main FluctuatingFinite Element Analysis -ASimulationPackage forBiomolecular SimulationofElectron Microscopy Data Base (EMDB) Structures

Albert Solernou,Ben Hanson,Glenn Carrington,Rob Welchand Sarah Harris

Overview We have developedanew software tool forbiomolecular simulationknown as Fluctuating Finite ElementAnalysis(FFEA),which is specifically designed to take advantageofthe current explosion in thequantity andquality of structural data fromcryo-Electron Microscopy (cryoEM) beingdeposited in theElectronMicroscopy Data Base (EMDB).Atthe atomistic level, MolecularDynamics(MD)simulations have been widely adopted by thebiomolecular sciences community to calculate proteindynamicstrajectoriesfromthe structural information availableintheProteinData Base (PDB). FFEA aims to provideanequivalenttoolfor protein simulationbut using structures fromthe EMDB,rather than thePDB.

FFEA simulates thedynamic behaviourofcollections of proteins or largeproteincomplexes. It provides access to theelusive mesoscale regime (between 10 and500nm)thatistoo computationally expensivefor atomisticMD, buttoo smallfor conventional macroscopic simulationtechniques.FFEAproducesatrajectory that is analogous to aconventional atomistic (orcoarse-grained) MD simulation, only thedynamicsofthe macromolecule are calculated on amesh usingstochasticpartial differentialequations derived fromcontinuum mechanics. FFEA treats asoftmacromolecule as acontinuum viscoelastic solid that is subject to thermalnoise, whichmust be chosen so as to satisfythe fluctuation-dissipation theorem. Thematerialpropertiesofthe proteinare determined by thedensity,Young’s modulus and internalviscosity.The motion of thecontinuum is governed by thestochasticpartial differential equation: where u LV WKHYHORFLW\ȡLV WKHGHQVLW\ ı is thecontinuum Du stress, f arethe body arising fromthe material U ı f ˜’˜’ ʌ deformationand external interactions;and ʌ is the Dt stochasticstress arising from thermalnoise.

This equation,togetherwiththe material constitutiveequationfor thecontinuum stress,is discretisedusing thefiniteelementmethodbysubdividing theprotein into aset of tetrahedral elements(seeFig 1).ThisyieldsasystemofLangevin equationsdescribingthe motionofthe element nodes.The method has no upperlength-scale(andconverges smoothly to the athermallimit at largelengths),however thecontinuumapproximationbreaksdownfor mesh element sizes<5Å.The flexibilityofthe simulatedproteins is determined by theYoung’s modulus, whichisusually obtainedbycomparingthe rangeofconformersobtained in the FFEA simulations with negative stainEMimages.

To describeensembles of interactingproteins, FFEA usesshort-rangevan derWaals forces. Thesepreventstericoverlap,and can also includeareversible attractive interaction over longer length-scales. Separatebiomoleculescan also be permanentlytetheredbyharmonic springs,and external forces can be appliedtoindividualnodes of theFEmesh.The of thesurroundingsolvent in FFEA is represented as simple Browniannoise, andweare currentlyimplementing theabilitytorepresent solvent flow. FFEA is shortly to be released as open-sourcesoftware formesoscale biomolecular simulation. Thesoftware is stored in theBitBucket repository,which facilitates versioncontrol during development.Ithas users’ and developers’ manuals, atutorialguide fornew users,a documented visualisationtoolwhich is apluginfor theestablishedPyMolprogram,and a robust test suite to ensure that thephysics within FFEA is workingcorrectly.The software is availablefromthe Bitbucketsoftware repository on request.

31 main

Figure 1: FFEA simulationscytoplasmic dynein on amicrotubule track.To investigatethe importanceofthe interaction between thetwo monomers during walking, in ourmodelsthey are connectedtogether by springs(shown in grey)ofvarying lengthsand stiffness.

We are currently using FFEA to modelthe mechanismofactionofflagellarand cytoplasmic , fibrinogen aggregationduringblood clotting, ,and theinner kinetochore.With theInstitute forProteinResearch in Osaka, Japan,weare developing methodsto parameteriseFFEA simulations from atomistic models, andexperimentaldataon biomolecularflexibility. By developing anew mesoscale modelofsoftmacromolecules, we aimtoopenupnew regimesofsoftmatterphysics and molecularbiology to computer simulation. Ourgoalisthatthe FFEA methodologywillbecome astandardtoolfor in silico design andinvestigationatthe mesoscaleinboththe commercialand academicsectors. Since FFEA bridgesthe gapbetween atomistic andconventionalFiniteElement modelsof biological organs,blood flow andtumourgrowth, andtherefore facilitates thefuture developmentofmulti-scale simulationschemesthatoperate from theatomistic throughtothe macroscale.

Collaborators University of Leeds: D. Read and O. Harlen (School of Mathematics).

www.astbury.leeds.ac.uk 32 main Towards abetterunderstanding of oxidativebiomassdeconstruction

Glyn Hemsworth

Introduction Thebreakdown of waste plantmaterial, in theformoflignocellulose,for subsequent fermentation into biofuels holdstremendouspromise forsecuring humankind'sfuture energy needs. Though research over severaldecades hasprovided many details of howfungi and microbes areabletodegrade thesematerials,the industrial production of lignocellulosic biofuels hasremainedtoo costly to competewithfossilfuels.The discovery of lytic polysaccharidemonooxygenases(LPMOs) in 2010/11was, therefore, akey breakthrough. Theseenzymes oxidativelyinducechain breaks into thecrystallineregionofhardtodegrade polysaccharides such as cellulose andchitin, therebyaugmenting theabilityofotherenzymes to degrade biomass. Indeed, theinclusionofLPMOs in enzymatic cocktails forcellulose degradation provides considerable improvements in theefficiencyofsaccharification, so there is nowaworldwidedrivetoensure that theseenzymes areusedeffectivelyinindustry. Our research is focused on theelectron transfer processes that mightbeusedtoactivate LPMOs.

Results CAZy(www.CAZy.org)isacomprehensive database of structurally-related, catalytic andnon- catalytic modulesfromenzymes involved in thedegradation, modification or synthesisof carbohydrates.Using this resourcewehaveidentified arange of target proteins that contain domainsofunknown funtion, denotedX-domains, appended to carbohydratebinding modules. Sequence analysis of theseX-domains suggeststhattheymay well play an electron transferfunctionand so we have selected asubsetoftargets to be characterised(Figure 1A).

Figure 1: (A) thedomain architecture of target proteins to be characterised. (B) purification gels showing progressonthe isolationof individual X-domainsfor characterisation.

We arecurrently workingonexpressing andpurifying thesedomains (Figure1B) with theaim of combiningstructural studiestogetherwithbiochemical, spectroscopic andredox analyses to gain athoroughunderstanding of X-domain function.Wewill then seek to exploitthe knowledgegainedthrough enzymeengineeringtoensuremaximal enzymeturnover during biomass degradationwhilstalsoinvestigatingother potentialapplicationsfor thesedomains.

Funding This work is funded by theBBSRC.

Collaborators University of Leeds: A. Berry External: B. Henrissat (AIX Marseille Université),P.Walton(University of York), A. Parkin (University of York).

33 main Production,purificationand characterization of proteins of the proteobacterial acinetobacterchlorhexidine efflux, PACE family involved in antimicrobial resistance

IrshadAhmad, Karl Hassan,Scott Jackson,Ian Paulsen andPeterHenderson

Introduction Drug resistance is an increasing problem in clinical settings with some bacterialpathogens nowresistanttovirtually allavailable drugs.Chlorhexidine is abisbiguanideantimicrobial agentthatisextensively usedinantiseptics rangingthrough soaps,mouthwashes and preservatives. Increasing resistance to chlorhexidineisseeninsomepathogens such as Acinetobacter baumannii,inwhich anovel efflux resistance wasassociated with agene designated aceI (for AcinetobacterchlorhexidineeffluxI). We have cloned genes encoding proteins that arehomologues of AceI,withaviewtotheir amplifiedexpression,purification andcharacterizationasapreliminarytowards structural studies.

Results GenesencodingAceIhomologues from23species of bacteria were transferred to the pTTQ18 plasmid vector,and transformed into Escherichiacoli BL21(DE3)hostcells,where theexpression of each cloned gene in membrane fractionswas detected in Coomassie stainedSDS gels comparingpreparationsfrominduced with uninducedcells (nineexamples are shown in Figure 1).Westernblots detectingthe His6-terminus of each proteinwereused to verify theextentofexpression (Figure1).

Figure 1: expression screeningofPACE family proteins. SDS-PAGE(top) andWestern blot (bottom) analysis of membrane preparations fromsmall-scaleculturesofE. coli expressing each indicatedprotein. Lane 1 shows molecular weight markers for un-induced (UI) and induced (I)preparations asshown.

Out of twenty threeinvestigated,seven geneswere expressedatlevelssufficientfor productionata30 litre fermentationscale. Each of thesewas then purified in mg quantities by IMAC.The appearanceofmorethanone band (cfFigure2)doesnot necessarily represent degradationormultimers,but an effect of SDS in partially unfoldingthe moleculesina preparation.The integrityofthe purified proteinswas also assessed by assaying bindingto knownorputativesubstrates(Figure 3a). Several of thehighlyexpressed AceI homologues conferred resistance to acriflavine, anucleic acidintercalatingbiocide.Acriflavine fluorescence is reducedwhenitisintercalated in nucleicacids,allowing real time measurementsofacriflavine transportinE. coli cells(Figure 3b), showingactivityofthe over- expressed proteins.

www.astbury.leeds.ac.uk 34 main

A 1 2 3 4 5 6 B 1234 6 66 5 76 45 52 36 38 31 29 24 24 17 20 12 14

Figure 2: purificationofFbal3166protein.Fbal_3166protein waspurified from theinner membrane of E. coli BL21(DE3) pTTQ18 (Fbal_3166)-His6. Theinner membranesweresolubilisedin1%DDM.(A). Coomassie blue stained 15% SDS-PAGEofFbal_3166 protein(B). Westernblot. Samplesloaded as follows: (1) mol. Wt. markers(kDa); (2)membranes; (3) supernatant;(4) membrane pellet;(5) unbound flow;(6) purifiedprotein.

3.0 Chlo rhexidine on (% ) 40 Spermidine ti 2.5

Cadaverin e or

30 Putrescine op 2.0 uench pr

eq 1.5 20 nc 1.0 sce 10 escence re 0.5 uor uo Fl Fl 0 0.0 010203040 0100 200 300 400 [Ligands](µM) Time (Sec)

Figure 3: activities of theFbal_3166 protein.(A)(Fbal3166)-His6 proteinwas purified fromthe inner membrane of 30L cultures of induced E. coli BL21(DE3) pTTQ18-Fbal3166)-His6,and solubilisedin1% DDM. Thefluorescencechange(quench) of tryptophan residues in theprotein wasmonitored atincreasing concentrationsofthe potential ligands:chlorhexidine (blue);spermidine(red);cadaverine (green);putrescine (mauve).(B)Small volumecultures were grownofcells expressingthe Amva chlorhexidineeffluxprotein (green),induced cellsexpressingFba-His6protein (blue),uninduced cellsnot expressing theFba3166 protein (black),and cellscontaining pTTQ18 plasmidwithout thegeneinsert(red).D-glucose wasadded at thepoint indicatedbyanarrow in order to energise efflux of fluorescent acriflavine. Cells expressing FbaHis6are competentfor efflux of acriflavine.

Currentworkcontinues with theseven homologuesofthe PACEefflux transporter family that have been purified andtheir abilitytobindand transport ligandstested.Structural studiesare underway.

Funding This work is funded by ProjectGrantsGNT1060895and GNT1120298 fromthe NHMRC (Australia)toIP, KAHand PJFH.PJFH thanks theLeverhulmeTrust foranEmeritus Research Fellowshipand KAHthanksthe EU foraMarie Slowdoska CurieResearchFellowship.

35 main An integrated approach to the study of cellular interactions with amyloid

MatthewJackson,Clare Pashley, Sheena Radford and Eric Hewitt

Introduction Theformation of insolubleamyloid fibrils is associated with aspectrumofhuman disorders, the amyloidoses, whichinclude Alzheimer’s, Parkinson’s, type 2diabetes anddialysisrelated amyloidosis (DRA).Inthese disordersthe formationofamyloid fibrils is associated with cellular dysfunctionand tissue destruction.Yet despite decades of researchthe culprit speciesand mechanisms of amyloidtoxicityremainpoorlyunderstood.

Ourgoalistodetermine how thestructure andphysical propertiesofamyloid affectscellular physiology andviability.This involves amultidisciplinaryapproachinwhich information obtained by NMR, atomicforce microscopy,electronmicroscopy,photo-crosslinking,mass spectrometryand fluorescence basedspectroscopictechniques is integratedwithanalyses of cellfunctionand viability. We are studying theoligomeric assemblyintermediates, fibrils and fibril-derived oligomersformedbyanarrayofamyloidogenic precursors,including Į-synuclein (Parkinson’s),amyloid-ȕ (Alzheimer’s) and ȕ2-microglobulin (DRA).Experimentalapproaches usedtoanalyse theinteractions andeffectsofthese amyloid species on cells includeplate- based assays forcellviabilityand ,livecellconfocalmicroscopy microscopy,flow cytometry,subcellularfractionationand proteomics. In addition,weare exploringapproaches forthe delivery of amyloidaggregates into thecytoplasmofsingle cells with colleaguesinthe SchoolsofBiomedical Sciences andElectrical andElectronic Engineering.

Figure 1: imagingofcellular interactions with Į-synuclein fibrils. SH-SY5Y cells were incubated with different OHQJWKV RI Į-synucleinfibrils labelled with thefluorescent dyetetramethylrhodamine (TAMRA) and lysosomes stained with LysoTracker dye.

Publications Jackson M.P. &HewittE.W.(2016)Cellularproteostasis: degradation of misfolded proteins by lysosomes. EssaysBiochem. 60:173-180.

PashleyC.L., Hewitt E.W. &Radford S.E. (2016) Comparison of theaggregationof homologous beta(2)-microglobulin variants reveals protein solubilityasakeydeterminant of amyloid formation. J. Mol. Biol. 428:631-643.

Funding This work wasfunded by theWellcomeTrust,EPSRC andERC.

www.astbury.leeds.ac.uk 36 main Amodelmembrane system to mimicfolded cristae of mitochondria andthylakoid stacksofchloroplast

George Heath, MengqiuLiand Lars Jeuken

Introduction Multilayered lipid membrane assembliesare utilized throughoutnatureand are involved in a widerange of energy producing pathways,fromthe double membranes surrounding mitochondria andgram-negative bacteriatothe stacked thylakoidmembranes of photosyntheticplant chloroplasts.The benefits of multiplemembranestackslie with theability to amplifyand compartmentalizesinglemembrane functionsinseries. This presents significanttechnological potential formimicking andharnessing adiverse rangeofenergy productionmachinery.Such applications rangefromproducing fuels fromorganicmatter, to convertingorganicmatterinto electricity andexploitingphotosynthesisfor solar power. In addition to energy productionithas been thoughtthatmultilayered membranes mayallow the serialcouplingofmembrane proteinsthatcould lead to newapplications in photonics, biosensing and the3Dcrystallizationofmembraneproteins. Accompanyingthe functionality, themultilayered structures also offerincreased mechanical robustness,atraitthatcan hinder single membrane systems. To utilisethe powerofmultimembrane stacks in biotechnology, methodologiesare requiredtocreate thesesystems in asimple, stable andscalable manner. Theformation of multiplelipid membrane stacks hasbeenpreviously demonstrated by a number of groups, buttheir constructionseitherdoesnot allowthe incorporationofmembrane proteins or thesystems requirelaborious procedures or even custom-synthesised lipid analogues. Here we describe atechnique using poly-l-lysine (PLL)asanelectrostatic linking polymerlayer between membranes.PLL is awidelyusedbiocompatible cationicpolypeptide that is inexpensive, biocompatable and readily available.

Results Assemblyofthe PLLmultilayered lipidbilayers is schematically shown in Figure 1(Top).PLL is allowedtobindtoanegatively charged ‘base’SLB (supported lipid bilayer) (POPC/POPG 1:1) untilsaturation. Excess PLListhenrinsedawayand asecond membrane is formedon topvia asolutionofnegativelycharged vesicles (POPC/POPG 3:1). This process is then repeated foreachadditional layer: bindingofPLL followedbyrupturing vesicles in alayer-by- layerfashion.Whilstthe formation of thefirst ‘base’ bilayerrequirescalcium in solutionas ‘fusagen’,all subsequent lipidbilayersdonot,since they bind to thepositivelycharged PLL layer. Theformation of themultilayerstack wasconfirmedbyfluorescence microscopy,quartz- crystalmicroballance with dissipation(QCM-D) andatomic formce microscopy(AFM).The AFMresultsindicatesthatwhenthe stacks getmore than three membrane layers, they become very heterogeneous with patchesofmembranesinthetensof nanometerrange.The ‘patchy’nature of themembranesinthemultilayerstacks wasconsistentwiththe QCM-Ddata, whichshowedthe dissipationincreasing witheach additionalmembranelayer. To studyfundamental biological interactionsand forfuture potential technological applications in multiple membrane systems,the ability to incorporate membraneproteins is vital. Figure 1: aschematicrepresentationofthe formationof Thus we followedthe same method to amultilayer membrane stackusinglayer-by-layer assembly of negatively charged lipid vesicles that ruptureonthe surface to form planar membranes and positively chargedPLL.

37 main form amembrane stack, butformedthe second lipid bilayerwithproteoliposomes (POPC/POPG A D 3:1) containingatransmembraneprotein.Intotal three membrane proteins were investigated: two differentdrug-metabolizingmono-oxygenases, humanflavin-containingmonooxygenase 3 (hFMO3)and cytochrome P450 2D6(CYP2D6). Thethird proteinstudiedwas theheterotrimeric B E outermembraneproteincomplex MtrCAB which playsacentralroleinthe metalreducing abilityof Shewanella oneidensis MR-1.Wetypically used proteoliposomes with 1-2% proteintolipid ratio (w/w). Foreachprotein,AFM images were obtained of thebasebilayer (1:1 POPC/POPG) C before incubationwithPLL andsubsequent F secondary bilayerformation withproteoliposomes. AFMimagesshowedalmost complete2nd bilayer coveragewithmanysmallprotrusions corresponding to extra-membranousregions of the proteins Figure 2: thethree proteindoublebilayer assemblieswereinvestigated using By labeling each proteinwithafluorescentdye,the fluorescencerecovery afterphotobleaching diffusion properties were investigated firstlyina (FRAP). (A-C)showsfluorescent microscopy single base bilayeralone andtheninthe second images after labelling proteins(hFMO3, CYP2D6 and MtrCAB, resp.) with Texasred bilayerofadouble membrane.Figure 3, A-Cshow (MtrCAB) and fluorescein (hFMO3 and double bilayers containingeachlabeledprotein in CYP2D6) dyes.Plots (D-F)show thesecondbilayer immediatelyafter bleaching a fluorescencerecovery after bleaching forthe centralregion. Therecovery of thephotobleached threeproteinsindouble bilayerswithfitsto single (blue dashed line) and double (red solid area (FigureD-F)indicates that allthree line) exponentials. membrane proteins freely diffuse within the membrane stack.

This newmultilayermembranemodel system opensuppossibilities forrecreatingand amplifyingsomeofnature’smostusefulmachineries. This work is also akey component in thedevelopmentofartificial gram-negativebacterialdoublemembraneswiththe intension of understandingelectron transport across multiplemembranes viaanumberofmetalloproteins. As such it notonly provides asystemfor futurepossibletechnologies butalsofor understandingfundamental biological processes at membrane-membraneinterfaces.

Publications HeathG.R., Li M.,Polignano I.L.,Richens J.L.,CatucciG., O'Shea P.,Sadeghi S.J.,Gilardi G.,ButtJ.N.&JeukenL.J.C.(2016)Layer-by-layerassembly of supportedlipid bilayerpoly- L-lysine multilayers. Biomacromolecules 17:324-335.

Funding This work wasfunded by theBBSRC.

Collaborators External:I.Polignano,G.Catucci,S.Sadeghi,G.Gilardi (University of Torino, Italy);J. Richens, P. O’Shea (University of Nottingham,UK; nowinthe University of BritishColumbia, Vancouver, Canada); J. Butt (Universtiy of East Anglia,Norwich, UK).

www.astbury.leeds.ac.uk 38 main Optimalreactioncoordinates

PolinaBanushkina andSergei Krivov

Introduction Advancesincomputerhardware andsimulationmethodologyhavemaderealistic simulations of complexbiological systemspossible. Nonetheless,because of thehigh-dimensionality of theresultingtimeseries, thegeneration of many trajectories perseis notsufficienttoprovide full scientificinsight. Eventually it becomes necessary to synthesizethe data into as faithful as possibleapictureofthe process of interest. Giventhe growingsizeand complexity of simulations, analysis andinterpretationofsuchdataare widely recognized as fundamental bottlenecks in theapplicationofatomistic simulations. Acommonway to analyze asimulation is to determinethe freeenergylandscape,i.e., thefreeenergyasafunction of oneormore reaction coordinates.Inspite of itsfundamental importancethe systematicresearch into how properlyselect such reaction coordinatesisstill in itsearly stages.Herewesuggest the definition of theoptimal reaction coordinatesand derive their important properties.

Results Thedynamicsofcomplex molecularsystems (e.g., proteins)isoftenanalyzedbyprojection onto areactioncoordinate (RC).The dynamics is then describedinasimpleand intuitive way as diffusion on theassociated free-energy profile. However, this simple diffusive description is notcorrect becouse thedynamicsprojectedonacoordinateisgenerally non-Markovianand subdiffusive.While,inprinciple, thegeneralized Langevin equationcan be usedtodescribe thenon-Markoviandynamicsexactly,however determinationofits kernel is very difficult.

Theframeworkofoptimal RCs employsanalternative strategy.Itselects RCs in an optimal way, i.e., to make theprojected dynamics more diffusive,tominimizenon-Markovianeffects, or to computeexactly aparticular dynamic property. While,ingeneral,the non-Markovian effectscannotbeeliminatedcompletely, theprojected dynamics could be modeledwithgood accuracy as diffusive or Markovian. In particular,some quantities can be computedexactly for theoriginaldynamicsonamultidimensional free-energy landscape of anycomplexity.

In adescriptionofreactiondynamics(i.e.,the dynamics betweentwo endstates),the following quantities areofparticular interest: thereactionflux, themeanfirst passage times(mfpt), and themeantransitionpathtimes (mtpt).While,inprinciple, onemay expect adifferentoptimal RC foreachofthe quantities, thecommittor RC canbeusedtocompute allofthemexactly. Also, theequilibriummeansquared displacement growslinearly with time as forsimple diffusion.The committor equals theprobabilityfor thetrajectory to reachone boundarystate (e.g., thenativestate in theanalysisofprotein folding) before it reaches another (e.g., the denatured state) starting from anygiven configuration.

TheequilibriumfluxJbetweenthe boundary statesofthe committorcan be computed exactly 1 1  dq as /)( kTqF ,where F(q) and D(q) are thefreeenergyand thediffusion ³³dqeJ /)( kTqF 0 0 qDe )( coefficient as functionsofthe committor (q).Corrspodingly,the mfpt,can be computed exactly 1   as mfpt /1 ³ eq (( 1) dqqqPJ /1) Jq .The mtpt,can be computed exactly as 0 1   mtpt /1 ³ eq 1()( dqqqqPJ 1() /) Jqq .One can take anytwo points on thecommittor 0

(q0 and q1)and use them as boundaries that define twonew endstates: q

39 main c  by simple rescaling, /()( qqqqq 010 ) ,which meansthatthe abovequantities can be computed exactly betweenany twopoints on thecommittor, notjust theboundary nodes. For themeansquared displacement of thecommittor onefinds '' 2 2)( ' tJtq ,i.e., the diffusive behavior.

Thus,while generallythe dynamics projectedonthe committor is notexactly diffusive and Markovian, in many respects it behavesasitisdiffusive.One maycompute some important properties exactly by using thesimplediffusive modelwiththe free energy profile andthe diffusion coefficient as functions of this optimalRC. Consequently,these free energy profile anddiffusion coefficientcan be used, in particular,torigorouslydefinethe free-energy barrier andpreexponentialfactor-othermajor descriptors of reaction dynamics, andingeneral,to make possiblearigorous analysis of complexbiological dynamics.

To applythese resultsinpractice, oneneedsefficientand robust methodstodetermine the optimalRCfromatomistic simulations,which is currentlyawork in progress. Anumberof approacheshavebeensuggested,which can be usedtodetermine an optimizedRC, which is asignificantimprovement over conventionallyemployedsimpleRCs,but is stillless optimal

Figure 1: freeenergy landscapeofHP35 Nle/Nle mutant as afunctionofaputative optimalreaction coordinate, rescaled so that thediffusion coefficient is D(x)=1. Themeanfirst passagetime, computed fromthe diffusive model is twotimes shorterthan that computed directlyfromthe simulation trajectories, meaning that thecoordinateisclose to thecommittorbut not yetequal to it.The representativestructuresfor theregionsofthe landscape show atrajectory snapshotclosestto theaverage structureofthe region. Colour code the root-mean-square (rms)fluctuations of atomic positions around the averagestructure. than thecommittor(Fig. 1). In ourlab,weare currently developinganapproachwhich, given alongtrajectory, will determinewithhighaccuracy theoptimal RCsand theassociated free energy landscape with minimal inputfromthe user.

Publications Banushkina P.V. &KrivovS.V.(2016)Optimal reactioncoordinates. WileyInterdiscip. Rev.- Comput.Mol.Sci. 6:748-763.

Funding This work wasfundedbythe BBSRC.

www.astbury.leeds.ac.uk 40 main Receptor tyrosine kinase signallinginthe absenceofgrowthfactorstimulation

EleanorCawthorne,Janne Darell, Christopher Jones, Sabine Knapp, Chi-Chuan Lin, Dovile Milonaityte, ArndtRohwedder,CarolineSeilerand John Ladbury

Introduction In theabsence of extracellularstimulationorgenetic mutation, an oncogenicresponse can be drivenbythe competitivebindingofSH3 domain-containingdownstreameffectorproteinsto proline-rich sequencesongrowthfactorreceptors. Of theapproximately50plasmamembrane receptortyrosine kinases(RTKs)the majority have proline-rich sequencesintheir C-termini. Thesehaveapropensitytobindtothe >300 proteins expressed in humancells whichcontain SH3domains. Theseinteractionsoccurinthe absenceofany extracellularstimulation(e.g. growth factors,cytokines). Proline-rich sequence binding to SH3domains are promiscuous andthe observed interactionswithRTKsare dependent on therelativeconcentrations of the proteins involved.

We previously establishedthatundernon-stimulatoryconditionsthe fibroblast growth factor receptor2(FGFR2)recruitsthe adaptor protein, growth factorreceptorbindingprotein2 (Grb2) through itsC-terminalSH3 domain. In cells depletedofGrb2 otherproteins canaccess theproline-ULFK PRWLIRQ)*)5 2QHRIWKHVH SURWHLQV SKRVSKROLSDVH & JDPPD  3OFȖ LV activated on bindingand through turnover of plasmamembranephospholipidstoproduce second messengers, raises cellularcalcium levels whichare responsible forincreased cell motilityand invasive behaviour.Inovarian andlungadenocarcinoma patients with lowlevels RI *UEDQG LQFUHDVHG H[SUHVVLRQ RI 3OFȖKLJKHULQFLGHQFH RI PHWDVWDVLVOHDGV WR JUHDWO\ reducedsurvivaloutcomes.

Results We have extended ourstudies in this area to explore other RTK-SH3 domain-containing proteininteractionstoestablishwhether theup-regulationofsignaltransductionthrough these interactions is ageneralphenomenon.Thisleads to thehypothesisthattwo tiers of intracellularsignallingcan be derivedfromreceptors with intrinsicproteinkinaseactivity: 1) Ligand-inducedelevationinkinaseactivityresultingintyrosylphosphate-mediated effector proteinrecruitmentand committal to adefined cellularoutcome (e.g.proliferation). 2) Receptor phosphorylation-independent activation of downstreameffectors through SH3 domain/proline-rich sequence interactions,which appear to be required forcell homeostasis/metabolic control.

Hyperactivityofthe tier 1signallingisafeature of receptor tyrosine kinase-relatedcancers arisingfromgenetic mutation. Althoughthe tier 2signallingmechanism occurs underbasal conditions, andisthuslikelytobeassociatedwithcellularmaintenance,wehaveshown that fluctuations in expressionlevelsofSH3-containingproteins can drivecells into pathological phenotypesincludingproliferation and metastasis.

We are testingthishypothesiswitharange of methods extendingfromcell-based assays (includingfluorescencelifetimeimaging microscopy) throughtostructural andinvitro biophysicalanalysis.

Focusingprimarily on gastro-intestinalcancers we have beguntoexplore theeffectsofstress on intracellular proteinexpressionand theoutcomesontier2signalling. We have shown that by mimickingconditionsexperienced in theGItract we can affect expression of receptor tyrosine kinases.

In addition to identifyingthe signallingpathwayswhich are initiated as aresultoffluctuations in proteinconcentrations in cell-based assays,weare exploring theinteractions associated withup-regulationofTier2signallingusing both biophysicaland structuralbiological methods.

41 main Highresolutionstructuraldetailonthe receptor-ligandinteractions areproviding invaluable detail on themodeofrecruitmentofsignallingproteinsaswellasinformation towards potential inhibition of aberrant pathways that lead to pathogenicoutcome.

Publications Kim J.Y.,Kinoshita M.,Kume S.,Hanke G.T., Sugiki T.,Ladbury J.E.,KojimaC., IkegamiT., Kurisu G.,GotoY.,etal. (2016) Non-covalent forcestunethe electron transfercomplex betweenferredoxinand sulfite reductase to optimize enzymatic activity. Biochem.J. 473:3837-3854.

BelousovaN., MikheevaG., XiongC.Y., StaggL.J., GageaM., FoxP.S.,Bassett R.L., LadburyJ.E., BraunM.B., Stehle T.,etal. (2016) Nativeand engineered tropismofvectors derivedfromarare species Dadenovirus serotype 43. Oncotarget 7:53414-53429.

Timsah Z.,Ahmed Z.,IvanC., BerroutJ., GageaM., Zhou Y., Pena G.N.A.,HuX., Vallien C., Kingsley C.V.,etal. (2016) Grb2 depletionunder non-stimulated conditionsinhibitsPTEN, promotes Akt-inducedtumor formation andcontributes to poor prognosisinovariancancer. Oncogene 35:2186-2196.

CampbellJ.C., KimJ.J., Li K.Y., Huang G.Y., RegerA.S., MatsudaS., Sankaran B.,LinkT.M., YuasaK., Ladbury J.E.,etal. (2016) Structural basisofcyclicnucleotide selectivityincGMP- dependent protein kinaseii. J. Biol. Chem. 291:5623-5633.

Carter B. Z.,Mak P. Y.,ChenY., MakD.H., Mu H.,Jacamo R.,RuvoloV., AroldS.T., Ladbury J. E.,Burks,J.K., et al. (2016) Anti-apoptotic ARC protein confers chemoresistanceby controlling leukemia-microenvironment interactioQV WKURXJKDQ1)ț%,/ȕVLJQDOLQJ QHWZRUN Oncotarget 7:20054-20067.

Funding This work wasfundedbythe Mathers Foundation,USA andthe NIH, USA.

Collaborators University of Leeds: P. Quirke,S.Short,A.Breezeand D. Tomlinson. External: Z. Ahmed;M.-C. Hung, S. Arur (UniversityofTexas MD AndersonCancerCenter, USA);M.Bogdanov (University of Texas, USA)and R. Grose (Barts Cancer Institute,London).

www.astbury.leeds.ac.uk 42 main Understandingthe requirementfor ionchannel activity during virus infection

Samantha Hover, BeckyFoster, Eleanor Todd,JohnBarr andJamel Mankouri

Overview SegmentednegativestrandedRNA virusescomprise several hundredspecies that are classified into Orthomyxoviridae,Arenaviridaeand Bunyaviridaefamilies. Thesefamilies contain adisproportionatenumberofserious humanpathogens includinginfluenza,Lassa andCrimean-Congo hemorrhagicfever viruses. Whilethese virusesare genetically diverse, allhavecommon structural features includinganouter host-cellderived envelope with inserted glycoproteinspikes, andsegmented RNA genomesthatare associatedwithmultipleviral proteins into ribonucleocapsid (RNP) complexes. TheseRNPsmust escapefromthe viral envelope in order to initiate infectionofsusceptible cells.Wehavepreviouslydemonstrated that Bunyamwera virus (BUNV), a model virus for the Bunyaviridae family, requires cellular potassium (K+)channel activityduringvirus infectionofmammalian andarthropod celllines. Subsequent analysis identified that K+ channelinhibitionwas detrimental to BUNVinfection during theearly post-entry stages of thevirus lifecycle, implicatingarole in virus-host membrane fusion andRNP release. We are nowusing BUNVtounderstandwhy K+ channel activitywould be required during viruslifecycles. We have produceddual-labelledfluorescent BUNVvirions to visualiseBUNVhostcellentry processes andfound that it trafficsthrough endosomal vesicles containingahigh [K+], whichisrequiredfor efficientvirus infection. BlockingK+channelswas foundtoshift endosomal[K+]withinendocytic vesicles fromlate endosomes into lysosomes indicatingK+channelsmay have functionswithinendosomal compartments,apreviouslyundescribed role.WebelievethatK+channelsare therefore actinginendosomal processestousurp theK+endosomalbalance requiredfor BUNVRNP release.

Publications HoverS., KingB., Hall B.,Loundras E.A.,TaqiH., Daly J.,DallasM., PeersC., Schnettler E., MckimmieC.,etal. (2016) Modulationofpotassium channelsinhibitsbunyavirus infection. J. Biol.Chem. 291:3411-3422.

Mankouri J.,WalterC., Stewart H., BenthamM., Park W.S.,Heo W.D., Fukuda M.,Griffin S. &HarrisM.(2016)Release of infectious hepatitiscvirusfromhuh7cells occurs viaatrans- golginetwork-to-endosome pathwayindependent of very-low-density lipoproteinsecretion. J. Virol. 90:7159-7170.

Mohl B.P.,BartlettC., Mankouri J. &HarrisM.(2016)Earlyeventsinthe generationof autophagosomesare required forthe formationofmembranestructures involved in hepatitis cvirus genome replication. J. Gen. Virol. 97:680-693.

SurteesR., Dowall S.D.,ShawA., ArmstrongS., Hewson R.,Carroll M.W.,MankouriJ., Edwards T.A.,Hiscox J.A. &BarrJ.N.(2016) Heat shock protein70family members interact withCrimean-Congo hemorrhagicfevervirus andHazara virusnucleocapsidproteins and performafunctional role in thenairovirus replication cycle. J. Virol. 90:9305-9316.

Funding This work wasfundedbythe RoyalSociety andthe British Lung foundation.

Collaborators University of Leeds: A. Whitehouse, A. Macdonald, A.Tuplin and J. Lippiat. External: A. Kohl, S.Goldsteinand S.Finke.

43 main An improved binary cloningsystem for studying the mechanistic biology of plants

Michael Watson,Yu-feiLin,Elizabeth Hollwey, Rachel Dodds, PeterMeyer andKenneth McDowall

Introduction Centraltothe studyand engineeringofplantsistheir transformation, whichisachieved most commonly using Agrobacteriumtumefaciens,the causal agent of crowngalls(or tumours)in dicotyledonous plants.The inductionofcrown gallsisinduced by thetransferofT-DNA, a segment of atumour-inducing plasmid that is resident in A. tumefaciens,into thenucleus of infected plantcells,wherein it is stably integrated into thegenomeand expressed. Thegenes required forthe transferofT-DNA have been identified andare well characterised.Moreover, thesegmentbetweenthe bordersofT-DNA can be engineered to contain heterologous DNA while maintaining theability to be transferredefficientlyinto cells.Indeed, A. tumefaciens is usedroutinely to transform many plantspecies of academic, agronomicaland horticultural importance. To ourknowledge,all of thesystems that have been developed forthispurpose incorporated binary vectorsthatallowpropagationinEscherichiacoli,wherein DNAcan be readily cloned andmanipulated betweenthe bordersofthe T-DNA, priortotransferinto A. tumefaciens andfinally plants. Forany functional studyitisofcourseimperativethatthe integrity of constructs carrying insertsbetween theborders of theT-DNA is maintained.

Results Recently,aspartofastructure-function studyofArabidopsis thaliana MET1,which encodes acytosine-DNA-methyltransferase involved in epigenetic gene regulation, we observed that E. coli cells containing aconstruct encodingMET1producedunusually smallcoloniesonagar plates andwere extremely difficulttopassage,i.e.subculture,inliquidmedia (Figure 1). Moreover, this poor growthprovidedsufficient selective pressure for mutants, some of which hadrearrangementsofthe plasmid,todominatethe populationwhendense cultures were eventuallyobtained. Theadverseeffect of pGreenIIonE. coli growth wasalso evidencedby extensive filamentation (i.e.incomplete septation) of cells (Figure 1).

Figure 1: JURZWK RI '+Į FHOOV FRQWDLQLQJS(7D D VWDQGDUG H[SUHVVLRQ YHFWRU  S*UHHQ,, DQdpMET1-03 (pGreenIIwithMET1insert). (A) Cellmorphology. (B) ThegrowthofE. coli usingovernight cultures as an inoculum. Thedata-pointsfor pET28a andpGreenII are represented by blue diamondsand redsquares, respectively. (C) Colony morphology. (D) RE analysisofaselection of plasmidsisolatedfrom spontaneous mutantsthatproduce largecolonies. Labels that areoutlinedindicate plasmids with obvious rearrangements.

www.astbury.leeds.ac.uk 44 main Thefindingthataplasmid-based construct can affect thegrowthofE. coli wasnot in itself unusual.However, closerinvestigationrevealed that thevector(pGreenII), oneofthe most widelyusedbinaryvectors in Agrobacterium-mediated transformation,was itselfwithout any insert affectingthe growthofE. coli substantially, whichinturn placed pGreenII-based constructsunder considerable selectivepressure.Tominimisethe risk of constructs destined forplantsbeing mutated, we have produced, through aseriesofgenetic screens, anew versionofpGreenII that does not affect thegrowth of E. coli (Figure2).

Figure 2: characterisation of pViridis and an intermediateinitsproduction, pGreenII-IS5. (A) Theregion EHWZHHQWKH &RO( RULJLQ RI UHSOLFDWLRQ DQGWKH ƍ HQG RI npt1 (conferskanamycin resistance)inpGreenII-IS5. Shownare thepositions of an IS5element that wasintegrated into pGreenII and the“Bal31deletion” that was final step in theproductionofpViridis. (B) Cell morphology. (C) Growth in liquidculture using overnight cultures as an inoculum.The triangles andcirclescorrespond to data-pointsfor cellscontaining pViridis and pGreenII-IS5, respectively.Comparewiththe diamondsand squares correspond to data-pointsfor cells containing pET28a andpGreenII, respectively,inFig. 1.

We have also selectedanewstrainofE. coli that bettertoleratesexistingpGreenII-based constructswithout reducing theyield of plasmid recovery(data notshown). Theadoptionof thenew derivativeofpGreenIIand E. coli strain,which we have namedpViridis andMW906 respectively,shouldminimisethe mutationofgenes destined forstudy in plants,while they are propagated andmanipulated in E. coli.Thisisturn should minimise thepossibilitythat unforeseenrearrangementcause themisinterpretationofmechanisticand other studiesin plants.

Publications Watson M.R.,Lin Y.F.,Hollwey E.,Dodds R.E.,Meyer P. &McDowallK.J.(2016)Animproved binary vector and Escherichia coli strain for Agrobacteriumtumefaciens-mediated plant transformation. G3-Genes GenomesGenet. 6:2195-2201.

Funding This work wasfundedbythe Leverhulme Trust.

45 main Electron microscopy of membrane proteins to underpinstructure guided inhibitor design

ShaunRawson,Emily Caseley, Paul Beales, Lin-HuaJiang,Colin Fishwickand Stephen Muench

Introduction Membraneproteinsare ubiquitous in biologyand arekey targetsfor therapeutic development. Despitethis, ourstructural understanding haslaggedbehind that of their soluble counterparts. This is primarilydue to thecomplications of expressing,stabilisingand crystallising membrane proteins. Therecent resurgence of electron microscopy (EM) andits requirementfor less homogenous protein and ahighlyorderedcrystallatticehas made it apowerfultechnique for studying membrane proteins. Ourgroup have been studying arange of membrane proteins through EM andhomology modellingtodrive structure based inhibitor design programs. However, anumberofproblems still remain,in-particular theabilitytostudy membrane proteins in more native environments.

Results Thegroup have been lookingatnew ways of studying membrane proteinsinmore“native” environments.The firstapproachhas been throughthe useofstyrenemaleicacid(SMA) co polymers, whichcut up themembraneinto lipid (SMALPs).Indoing so membrane proteins areenclosedbynativelipid,and showhigheractivitythantheir detergentsolubilized counterparts.Lastyearwepublishedthe firstnegativestain reconstructionofaSMALP extracted membraneproteinand this year have been workingonsingleparticlecryo-EM studiesinadditiontodevelopingnew polymers. In parallel, we have worked with theBeales grouptostudy newproteo-hybrid vesicles whichoffer extended functional lifetimes for membrane proteins.

Theoverallgoalofour studiesistofurther ourstructural andmechanical understanding of proteins andprotein complexestodrive thedesignofnovel modulators of function.Tothis endwehavearangeoftargetproteinswithinthe labincluding, V-ATPase,bc1,Enoyl Reductase, P2X7 andIGPD. We are combining electronmicroscopy, X-raycrystallography moleculardynamicssimulations andbiochemical techniques to providenew insights into structure function andinhibitor development(Figure1A).For thosesystems we have yetto resolveahigh resolutionstructure for we are using astructure modellingapproach with biochemicalvalidation. This is exemplifiedbyour work on P2X7,for whichwehavedeveloped aseriesofnovel andpotentinhibitors that both blockand openthe central pore based on a modelstructure.

Figure 1: (A) in silico designed smallmolecule inhibitorbased on modelledstructureofthe V-ATPase. (B) improvement in theV-ATPase EM reconstruction aftertaking intoaccount theinherent flexibility in thesystem. (C) directobservationofabound smallmolecule inhibitor within an EM reconstruction.

www.astbury.leeds.ac.uk 46 main Thegroup hasbeenusing EM in particular to studythe inherentflexibility within largebiological systems,for whichitiswellsuited(Figure 1B). Moreover, usingthe advancementsinEM we have been able to clearly resolvesmall molecule binding directly openingupthe door for structure guided inhibitordesignbyEMmethodologies (Figure1C).

Publications Rawson S.,Harrison M.A. &MuenchS.P.(2016)Rotatingwiththe brakesonand other unresolved features of thevacuolar ATPase. Biochem. Soc. Trans. 44:851-855.

Rawson S.,Iadanza M.G.,Ranson N.A. &MuenchS.P.(2016)Methods to accountfor movement andflexibility in cryo-EMdataprocessing. Methods 100:35-41.

LeeS.C., KnowlesT.J.,Postis V.L.G.,Jamshad M.,Parslow R.A.,Lin Y.P.,Goldman A., SridharP., OverduinM., MuenchS.P.,etal. (2016) Amethod fordetergent-freeisolation of membrane proteins in theirlocallipid environment. Nat. Protoc. 11:1149-1162.

Khan S.,LiM.Q., Muench S.P., JeukenL.J.C.&BealesP.A.(2016)Durableproteo-hybrid vesicles forthe extended functionallifetime of membrane proteins in bionanotechnology. Chem.Commun. 52:11020-11023.

CaseleyE.A., Muench S.P.,FishwickC.W.&JiangL.H.(2016)Structure-basedidentification andcharacterisationofstructurally novelhuman P2X7 receptor antagonists. Biochem. Pharmacol. 116:130-139.

Papachristos K.,MuenchS.P.&Paci E. (2016)Characterizationofthe flexibilityofthe peripheralstalk of prokaryotic rotary A-ATPasesbyatomisticsimulations. Proteins 84:1203- 1212.

MuenchS.P.(2016) Singleparticle cryo-EM, from sample to reconstructionintroduction. Methods 100:1-2.

ThompsonR.F., Walker M.,Siebert C.A., Muench S.P. &RansonN.A.(2016)Anintroduction to sample preparationand imagingbycryo-electron microscopy forstructural biology. Methods 100:3-15.

Funding This work wassupportedbythe MRC, Wellcome Trustand NIH.

Collaborators External: T. Dafforn (University of Birmingham) andR.McLeod(University of Chicago).

47 main Unified synthetic approaches to explore biologically-relevant chemical space

IgnacioColomer, Philip Craven,Richard Doveston,Christopher Empson,JamesFirth, DanielFoley,MatthewLilburn,ZachOwenand AdamNelson

Introduction Thediscovery of biologically-activesmall moleculesisanenduring themeinchemical biology andmedicinal chemistry.However,the historical exploration of chemical spacehas been highly uneven andunsystematic:asixthofknowncyclic organic compounds are based on just 30 (of2.5 million) known molecularscaffolds! This uneven exploration stems, in largepart, fromthe narrow toolkitofreliablereactions that currently underpinsmoleculardiscovery. We have establishedavibrant research programme focusing on thedevelopment of synthetic methods that have potentialtoexplore novelregions within chemical space. To assist prioritisation of methods able to explore chemicalspace appropriate forspecificdiscovery applications, we have recently launched theopen-access computationaltool, LLAMA(Lead Likeness AndMolecularAnalysis).

Abiosynthesis-inspired approach to naturalproduct-likescaffolds Asynthetic approach to diverse scaffoldswas developed that wasbroadlyinspired by terpene biosynthesis(Scheme).DielsAlder reactionswere exploitedtoconvert sp2-richsubstrates into complex three-dimensional intermediates.These intermediates were then transformed into 25 diverse andnovel scaffoldsusing cleavage,ringexpansion,annulationand rearrangementreactions.Itwas shown virtual libraries(enumeratedusing LLAMA)based on thescaffoldshad high naturalproduct-likenessand hadmolecularpropertiessuitable for applicationindrugdiscovery.

Scheme: syntheticapproachtonatural product-likemolecular scaffolds.

Summary Thedevelopmentofunified strategies that areabletodeliver skeletally diverse compounds withdefined molecularproperties is demanding. We have translated manyofour unified diversity-oriented syntheticapproachesintothe €196MEuropeanLeadFactory in which Leedsisapartner. Publications from this programme,and otherprogrammesunder active developmentinthe group, arelistedbelow. Furtherdetails of research withinthe Nelson groupmay be foundatwww.asn.leeds.ac.uk.

Publications StockwellJ., Daniels A.D.,Windle C.L.,Harman T.A.,Woodhall T.,LeblT., TrinhC.H., Mulholland K.,Pearson A.R.,Berry A.,etal. (2016) Evaluation of fluoropyruvate as nucleophile in reactionscatalysed by N-acetylneuraminicacidlyase variants: Scope, limitations and stereoselectivity. Org.Biomol.Chem. 14:105-112.

BurslemG.M., Kyle H.F., Breeze A.L., Edwards T.A., Nelson A.,WarrinerS.L.&Wilson A.J. (2016) Towards "bionic''proteins: replacement of continuoussequences from HIF-1D with

www.astbury.leeds.ac.uk 48 main proteomimeticstocreate functional p300 bindingHIF-1D mimics. Chem.Commun. 52:5421- 5424.

BurslemG.M., Kyle H.F.,Prabhakaran P., Breeze A.L.,EdwardsT.A., Warriner S.L.,Nelson A. &Wilson A.J. (2016) Synthesis of highly functionalized oligobenzamide proteomimetic foldamers by late stageintroduction of sensitive groups. Org. Biomol.Chem. 14:3782-3786.

Howard, J. K.,Müller, M.,Berry,A., Nelson,A.(2016)“An enantio-and diastereoselective chemoenzymatic synthesisofD-fluoro E-hydroxycarboxylic esters”, Angew. Chem.Int.Ed. 55,6767-6770.

Colomer I.,EmpsonC.J., Craven P., Owen Z., Doveston R.G.,ChurcherI., Marsden S.P. & Nelson A. (2016) Adivergent syntheticapproachtodiversemolecularscaffolds: Assessment of lead-likenessusing LLAMA, an open-access computational tool. Chem.Commun. 52:7209- 7212.

Firth J.D.,CravenP.G.E., LilburnM., Pahl A.,Marsden S.P. &Nelson A. (2016) A biosynthesis-inspired approach to over twenty diverse natural product-likescaffolds. Chem. Commun. 52:9837-9840.

FoleyD.J., Nelson A. &MarsdenS.P.(2016) Evaluatingnew chemistry to drivemolecular discovery:Fit forpurpose? Angew. Chem.-Int. Edit. 55:13650-13657.

Funding We thankEPSRC, BBSRC, theEUand GSKfor support.

Collaborators University of Leeds: S. Marsden External: I. Churcher, GSK; andA.Pahl(MaxPlanckInstitute forMolecularPhysiology). We also acknowledgeother scientificcollaborators whohavealsocontributedstronglytoother aspects of ouron-goingresearch programme.

49 main ABC-F proteins mediate antibiotic resistance through ribosomal protection

Liam Sharkey, Thomas Edwards andAlexO’Neill

Introduction Elucidation of themechanisms by whichbacteriaresistthe inhibitory effects of antibiotics provides essentialintelligenceinthe ongoingfight against antibioticresistance. Whilst the majorityofclinically important antibiotic resistance mechanisms arebynow well characterised, somekey gaps in ourknowledgeremain.One such gapconcernsthe mechanism by which ABC-F proteins mediate resistance to antibiotics that target proteinsynthesis in Gram-positive pathogens; although membersofthe ABC-F family collectivelyyield resistance to abroader rangeofclinically important antibioticclassesthanany other familyofresistance determinants, theirmechanism of actionhas been thesubject of controversy since theirdiscovery at the University of Leeds25years ago.

ABC-F proteinscompriseasingle polypeptidecontainingtwo ATP-bindingcassette (ABC) domainsseparatedbyalinkerof~80 aminoacids. In contrasttocanonical ABCtransporters, theABC portionsofABC-F proteins arenot fusedtotransmembrane domains(TMDs), nor are they genetically associatedwithTMDsinoperons.Two competing hypotheses have been proposedtoexplain howthese proteinsmediate resistance to antibiotics(Figure 1).The efflux hypothesis positsthatABC-F proteinsassociate with as-yet-unidentified TMDs to form a functionaleffluxcomplex capable of exportingantibiotics outofthe cell,whilstthe ribosomal protection hypothesissuggeststhatthese resistance proteinsact insteadtoreduce the accessibilityor affinity of antibiotic Figure 1: proposed mechanisms of ABC- bindingsites on the Fmediated antibiotic resistance. In an 50Ssubunitofthe antibiotic-susceptiblebacterium, translation is inhibitedbythe antibiotic, ribosome,thereby leading to growth arrest (centre). ABC-F directly protecting proteins (green)are proposedtorescue translation from inhibition either by thetranslational recruiting an unknownmembrane- machineryfrom spanningprotein to promote active efflux antibiotic-mediated of theantibiotic(left),or, by displacing the antibiotic fromits ribosomalbinding site inhibition.This (right). projectwas initiated withthe intention of elucidating themechanism of antibioticresistancemediated by ABC-Fproteins.

Results ABC-Fproteins protectstaphylococcaltranslationfromantibioticinhibition in vitro We initiallysoughttodeterminewhether ABC-F proteinscould mediate antibiotic resistance in vitro, underconditionswhere effluxwas notpossible. To this end, staphylococcalS30 extracts were generated, andusedtoestablish in vitro transcription-translation(T/T) assays. Twophylogenetically-distant ABC-Fproteins,Vga(A)and Lsa(A), were heterologously expressed in E. coli,purifiedtohomogeneity, andtested fortheir abilitytoprotectT/T assays fromantibiotic inhibition. Both proteins were shown to mediate dose-dependentrestoration of translationalactivity(Figure 2A). To provideconfirmation that theobservedability of ABC-F proteins to protect in vitro translation fromantibiotics reflectedthe activity of theseproteins in wholecells,we

www.astbury.leeds.ac.uk 50 main successfully recapitulatedinthe T/Tassaythree phenotypesthathavebeenassociated with theseproteins in bacteria.

ABC-Fproteins directlydisplace antibiotics from theribosome To assess whetherABC-F proteins protectthe translationapparatus fromantibiotic-mediatedinhibition by directly interferingwiththe interactionbetween theantibiotic andthe ribosome, we evaluated the abilityofthe Lsa(A)protein to 3 Figure 2: (A) titration of Vga(A) preventbindingofradiolabeled(H) (left)and Lsa(A) (right)intoT/T lincomycintopurified assays inhibited with a concentration of antibiotic staphylococcal ribosomes. Pre- sufficient to bring about90% incubation of Lsa(A)withribosomes inhibitionofthe system (IC90). priortothe additionof3H- (B) Lsa(A) causes adose- dependent reductioninbinding lincomycinresulted in adose- of radiolabelled lincomycinto dependent decrease in subsequent staphylococcalribosomes. bindingofthe drug to ribosomes (Figure2B). Subsequently,wealso examined theability of Lsa(A)todisplacepre-bound 3H-lincomycinfromribosomes, establishing that addition of an 8-foldmolar-excess of Lsa(A)toribosomes pre-incubatedwith drug resulted in asubstantial (~73%)reduction in ribosome-associated 3Hlincomycin. Collectively, ourresults show that theABCF-Fproteinsmediate antibiotic resistance through ribosomalprotection.

Amodelfor ABC-Fmediated ribosomalprotection Althoughthe moleculardetailunderlyingthisresistance mechanism is yettobeelucidated, we are able to propose amodel by whichABC-F proteins maymediate ribosomalprotection. This model is built upon therecent structural andfunctional characterisationofaninnate Escherichiacoli ABC-Fproteinthatdoes notconferantibiotic resistancebut insteadactsasa translationfactorthatrespondstothe ATP/ADP ratiowithinthe cell. This protein, EttA,binds theribosome at theexitsiteand insertsits interdomainlinkertowardsthe peptidyl-transferase centre (PTC), inhibiting translation when cellularenergylevelsare low. We propose that ABC- Fproteinsmediate antibiotic resistance by bindingtothe ribosome in thesamemodeasEttA, therebyallowingtheir interdomainlinker, whichisextendedby~30aa in comparisontoEttA, to contact theantibioticbindingsites at thePTC andcause either direct or allosteric displacement of thedrug. Supportfor this modelisfound in theobservationthatmutations withinthe linkerdomaincan alterthe resistance phenotypeofABC-F proteins. Future studies will focusongaining structural insights into theABC-F:ribosomecomplex.

Publications SharkeyL.K.R., EdwardsT.A. &O'Neill A.J. (2016) ABC-F proteinsmediate antibiotic resistance throughribosomal protection. mBio 7:e01975-15.

Funding This work wasfundedbythe BBSRC.

51 main Transcellular stress signalling: How proteotoxic stress is communicated between different tissuesinC.elegans

DanielO’Brien,Rebecca Aston, Vijay Shanmugiah,David Westhead and Patricijavan Oosten-Hawle

Introduction In allbiologicalsystems, cells throughout their lifetime are exposed to different physiological andenvironmental stress conditionsthatleadtoprotein damage andcellulardysfunction – and ultimatelydisease. Cumulativeproteinmisfoldingand aggregationisone of thehallmarks implicated in thepathologiesofmisfoldingdiseasesassociated with neurodegeneration, includingAlzheimer’s disease, amyotrophic lateral sclerosis, Huntington’s diseaseand Parkinson’s disease, as well as cancer, diabetes andseveral myopathies. Maintaining ahealthy cellularproteome is crucialtocellularviability. This is achievedbythe proteostasis network, whichintegrates protein biogenesis, protein foldingbymolecularchaperones, as well as clearancemechanisms andstressresponse pathways.The significance of cell protective Figure 1: modelexplainingtranscellular stress response mechanisms is nowwidely chaperonesignalling(TSC) in C. elegans appreciated to notjust actatthe levelofsingle cells,but at the“multicellular” level, throughout an entire organism. Thus,evolutionary conserved stress responsessuch as theheatshock response initiate inter-cellularcommunicationthatallows protective chaperone expression to be signalled andspreadfromone tissue to another, aprocess knownastranscellular chaperone signalling(TCS).One keyobservationofTCS is that an imbalanceofproteostasis in only onetissue(e.g. neurons) throughalteredexpression of themajor chaperonehsp90, can leadtoanupregulated hsp90 chaperone response in receiving tissuesthatisspread throughoutthe organism (Figure1). Howthese transcellularchaperone response mechanisms function at themolecular levelhowever remains an open question to date.

Results This project aims to determinehow transcellularchaperone signallingismediated between differenttissues. To address this question, we utilised asystems-wideapproach using RNA-Seqand further geneticanalysisofC. elegans strainswhere TCSisactivated through increasedhsp90 expressionin either neurons, intestine or bodywall muscle. This approach allowedustoidentify theGATAtranscription factor PQM-1asa mediator of transcellularchaperone signalinginC. elegans. Systemic depletion of pqm-1 by RNAi in thesestrainswhere TCSisactivated through increasedhsp90 expression,leads to reduced TCSthatis Figure 2: RNAi mediated knockdownofpqm-1 measured by the hsp90p::GFP reporter reduces TCS. pqm-1 RNAi in tissue-specificHSP90 expression level(Figure 2). WhilePQM-1 is overexpression strainsreduces transcellular knowntoberequiredfor lifespanextension hsp90p::GFP reporterexpression. Fluorescence in C. elegans,its precise role in the intensity is measured by ImageJ. regulationofproteostasisishowever unknown. Ourdatashows that C. elegans depletedfor pqm-1 exhibitincreasedsensitivity to heat stress,similartoanimals expressing mutant hsf-1,the master regulatorofstress

www.astbury.leeds.ac.uk 52 main responses (Figure3A).Furthermore,totestthe requirementofPQM-1 forthe maintenance of proteostasiswetookadvantageofC. elegans expressingQ35::YFPinthe bodywall muscle. Thesestrains areused as models forpolyQdiseases (e.g.Huntington’s Disease). Importantly, aggregationofQ35 is dependent on theefficiency of theproteostasisnetwork, thus these strainsalsoprovide ameasure for theefficiencyofthe proteostasisnetwork.Interestingly, the absence of PQM-1accelerates Q35aggregation by 1.5-fold, suggesting that PQM-1is required forthe maintenance of proteostasis(Figure 3B). Currenteffortsare aimedat elucidating howPQM-1 “signals”TCS from onetissue to another to modulate organismal proteostasis.

Figure 3: PQM-1isrequiredfor the maintenanceofproteostasis. (A) PQM-1is required forheatstresssurvival. Anull mutant of pqm-1 is sensitivetoheatstress, comparable to a hsf-1 mutant that cannot induce theheat shockresponse. (B) Q35 aggregationkineticsinC.elegans expressing Q35::YFP in thebodywall muscle. Depletion of pqm-1 leadstoincreased accumulation of Q35aggregates when compared to Q35 controlanimals. P=0.0001.

Publications O’BrienD.&van Oosten-Hawle P. (2016).Regulation of cellnonautonomous proteostasisin metazoans. Essays Biochem. 60:133-142.

Funding We aregrateful forsupport fromthe WellcomeTrust,the NC3Rs, andaBBSRC andMRC doctoral studentship.

Collaborators University of Leeds: D. Westhead.

53 main Proteins under the computational microscope: folding, disorder,mechanics,dynamicsand self-assembly

Emanuele Paci

Ourresearch focusesonthe development andapplicationofnovel computationaltools to investigate structure andfunctionaldynamicsofbiomolecules. Oneofour aims is fully exploit thewealthofstructuraldataobtainedwiththe advanced experimental probes availableat Astbury. Most of ourresearch involves experimentsperformedincollaborationwithcolleagues fromthe AstburyCentreand further afield.

Onetopic wheremodelsand simulations provided informationthatexperiments cannot provideand suggestionsfor more insightful experimentsrelatestothe structural propensity of polypeptides with specific sequence patterns. Theserange fromthe simplest repeat,a homopolypeptide, i.e.,repeatsofthe sameaminoacid,orrepeats short patternsofamino acids. While polymertheory suggests that most of thesesequences areunstructuredwefound evidenceofthe emergence of structuralpreferences andunique, sequenceand length dependent,dynamical properties. In collaboration with Michelle Peckham at Astburyand Ben SchulerinZurich we are exploringthe properties of polyampholyte repeat sequences that are strongly helicalusing FRET andsimulation.

Characterizationofdisorderedstatesisone of thecurrent focuses of ourgroup.Inthiscontext, in collaborationwithJaneClarke(Cambridge) we exploredthe role of an intrinsically disordered domaininthe foldingofSasG,aprotein that forms extendedfibrils in thesurface of Staphylococcus aureus andpromoteshostadherence andbiofilm formation(Gruszka2015 and2016). Much of ourworkinvolvesmolecularsimulation of thedynamicsand interactions withinand betweenprotein systems. Data generatedfromsimulationare used to interpret and direct experimental measurementssuchas hydrogen-deuteriumexchangeprobedbymass spectroscopy(in collaborationwithRoman Tuma), theresponse to mechanical of proteins that evolutionary adapted to function in extreme environments (Tyck2016).

Anotherchallengeundertakeninour group (in collaborationwithBruce Turnbull) is rational designofcapsids(forvarious practical purposes andtoassess ourunderstandingofthe forces involved in capsidformationand stability).We developed an originalapproachtoestimatethe stabilityand effectofmutations on capsids of knownstructure (a preliminarysteptothe design of novelcapsids) that hasbeenshown to be accurate in an applicationtolumazinesynthase, anon-viral protein that form capsids in several organisms(Hickman 2015). Figure 1: structural organizationofthe prokaryoticA-ATPase. (A) electron-microscopy In collaborationwithStephenMuench(Astbury) 3D maps of ion-pumpingrotaryATPases:V- we usedsimulation to characterizethe flexibility ATPase (left), A/V-ATPase(middle) andF- of theperipheralstalk of prokaryotic rotary A- ATPase (right). (B) twocopiesofthe peripheral stator EG complex(coloredinyellow and orange) ATPases. Structureshavebeen reportedfor the connectthe catalyticsubunits A/B (subunits peripheral stalk, thoughttorepresent colored in blue and red) with themembrane- conformationalstatesofthe stator during bound subunit I(colored in grey). (C) differentstagesofrotary catalysis.Weshowed superposition of thethree peripheral stator EG instead that thosestructures are conformers conformers PS1,PS2 andPS3.

www.astbury.leeds.ac.uk 54 main explored throughthermal fluctuations,but notstablestates. Thecoiledcoiltaildomainhas a high persistencelength(i.e., it is surprisingly rigid),but retains theability to adapt to different conformationalstates throughthe presence of twohinge regionsand accommodate structural changes in thecatalytic domainwhilstresistingthe largetorquegeneratedbycatalytic cycling. Theseresultsare importanttounderstandthe role thestators play in therotary-ATPase mechanism.

In ourgroup we also developand implement methods to determinefreeenergychanges along pathways connectingdifferent conformationalstatesofproteins.One particular topic on which we have been workingfor thepast20years is that of interpreting single molecule spectroscopyexperiments.Weare currently collaboratingwithDavid Brockwell to interpret measurementsofthe response to forceofthe transmembrane machinery that transports vitamin B12 andthe structural propensityofdisorderedregions of thesamemachineryfrom simulationand smallangle X-rayscattering data.

Publications Tych K.M.,BatchelorM., Hoffmann T., Wilson M.C.,HughesM.L., Paci E., BrockwellD.J.& Dougan L. (2016) Differential effectsofhydrophobic core packingresiduesfor thermodynamic andmechanical stability of ahyperthermophilic protein. Langmuir 32:7392-7402.

Tych K.M.,Batchelor M.,HoffmannT., Wilson M.C.,Paci E.,Brockwell D.J. &DouganL. (2016) Tuningprotein mechanicsthrough an ioniccluster graft fromanextremophilic protein. Soft Matter 12:2688-2699.

Papachristos K.,MuenchS.P.&Paci E. (2016) Characterizationofthe flexibilityofthe peripheral stalkofprokaryotic rotary A-ATPasesbyatomisticsimulations. Proteins 84:1203- 1212.

O'ConnorM., Paci E.,McIntosh-Smith S. &GlowackiD.R.(2016)Adaptivefreeenergy samplinginmultidimensionalcollectivevariablespace usingboxed moleculardynamics. FaradayDiscuss. 195:395-419.

Gruszka D.T., Mendonca C.,Paci E.,WhelanF., HawkheadJ., Potts J.R. &ClarkeJ.(2016) Disorderdrives cooperativefolding in amultidomain protein. Proc. Natl.Acad.Sci.USA. 113:11841-11846.

Collaborators UniversityofLeeds: B. Turnbull, D. Brockwell, L. Dougan, R. Tuma,S.Muench,M.Peckham. External: J. Clarke(Cambridge), A. Plückthun (Zurich), D. Glowacki(Bristol).

55 main How myosin 10 travels to filopodia tips

Ruth Hughes, Marcin Wolny, MatthewBatchelor, Francine Parker,Brendan Rogers,Glen Carrington,Marta Kurzawa, TomBaboolaland Michelle Peckham

Introduction Ourresearch interestsfocusonthe cytoskeletoninboth muscleand non-musclecells.This year,weour research hasrangedfrominvestigatingthe effectsofmutationsinthe Z-disc SURWHLQ Į-actinin in cardiacmusclecells to understanding howmyosin10trafficstothe filopodial tipinnon-musclecells.Wecontinuetodevelopminsuper-resolutionimaging,to imageproteinswithinprimary cilia in mammaliancells (withColin Johnson, in thefacultyof Medicine andHealth),recentlyfundedfromajointBBSRC-SFI grant. Acollaborationwiththe companyOrlaTechnologies, hasled to thedevelopment of novelsurfacesthatbetterpromote muscledifferentiationinculture.

Myosin10(Myo10) is part of themyosinsuper-family,which consistsof38genes,arranged into 12 classes.Myo10 is essentialfor theformationoffilopodia; thin actinrichprojections formed at thecelledge, whichcan act as pathfinders in migratingcells.Myo10 is importantfor both breastand prostatecancer metastasis. Ourquestionwas, howdoesthismyosinfindits waytothe tipoffilopodia?Toanswerthisquestion, we performedlivecelltotal internal reflectionmicroscopy (TIRFM) to imagefluorescentlytaggedMyo10,and determineits dynamics (incollaborationwithDrJustinMolloyand hisgroup at theCrick).

Results Ourimaging showedhow eGFP-Myo10 (Fig.1a) movestoand accumulatesatthe tipof filopodia(Fig. 1b,c). Singlemoleculescorresponding to eGFP-Myo10 were observed (Fig.

Figure 1: eGFP-Myosin10and itsbehaviourinlivecells. (A) eGFP-Myo10 construct (B) eGFP-Myo10 localises to thetipsoffilopodia(arrow)(confocalimage). (C) thefirst frame of aTIRFM image.Arrows show Myo10 at filopodial tips. (D) theboxed filopodiuminCis shownshows fluorescent spotswithinthe filopodium that correspondtosingle molecules of eGFP-myo10 (arrowedbelow). (E) Allthe fluorescent objectlocalisations obtained during avideo recording, used to generateasuper-resolution imageofthe paths takenbyeGFP- Myo10 within alivecell. Individual spot localisations (red) aresuperimposedoverthe averagefluorescence data(green). (F) akymograph of eGFP-Myo10 motility within afilopodium.The tracksshowamixtureof directed(d),stalled (s)and diffusive (df)motion. (G) Instantaneous of eGFP-Myo10 within a filopodium,showing twodistributions:one with an average instantaneousvelocity closetozero (stalledand diffusive behaviours) and theother with an average positive , showing directional(motorized) motion toward thefilopodial tip.

www.astbury.leeds.ac.uk 56 main 1d)and thelocalisations of thesemoleculeswere tracked.Asuper-resolution imageofeGFP- Myo10was generated by accumulatingall of thefluorescentobjectlocalisations obtained during avideo recording(Fig. 1e). Thediameterofthe filopodium wasestimatedfroman analysis of theaverage fluorescenceimage (shown in green) andthe estimate obtained by theindividualFL-M10spotlocalizations (red). Both approachesshowthatthe filopodial diameterestimated towards thebaseissimilar. However, this estimate starts to divergewithin theshaft of thefilopodium. Super-resolution data shows that FL-M10 moleculesare confined withinanaverage filopodial diameter of 116.5+/-18 nm (full-width at half maximum height) whereasthe diffractionlimited,averagefluorescence imagereports 357+/-28 nm (n=39). The super-resolutionimage additionally shows interestingsubfeatures in thesuper-resolved image. Theseincludeabulge on thesideofone filopodium andaconnectingbridgebetween twofilopodiaonthe upperright (yellowarrows).The image also showsshort tracksofeGFP- Myo10astheyaccumulate in thecellbodyjust before enteringthe filopodium.

Analysisofthemovement of eGFP-Myo10 withinthe filopodium demonstrates that themyosin exhibits three maintypes of movement:directedmotion(df), diffusive motion (d)and stalled motion(s) as themolecules move to the tip(Fig. 1f). Singleparticletrackingwas usedto quantify themotionofindividualparticles. Plottingthe instantaneous velocities showedthe complex behaviour of thesemolecules, in whichthe distribution required afit to twoGaussian functions. Onetypeofmotionhad an average instantaneous velocity close to zero (i.e.stalled and diffusive behaviors),the otherhad an averagepositive velocity,indicatingdirectional (motorized)motiontoward thefilopodial tip. Overall, ourwork demonstratedthatacombination of diffusiveand active movements is required to rapidly target myosin10tothe filopodial tip. Further work is underway to understand howthismotilitybehaviour isregulated.

Publications PeckhamM.(2016)How myosin organizationofthe actincytoskeletoncontributestothe cancer phenotype. Biochem. Soc. Trans. 44:1026-1034.

Parker F.,White K.,Phillips S. &Peckham M. (2016) Promotingdifferentiation of cultured myoblasts using biomimetic surfacesthatpresent alpha-laminin-2 peptides. Cytotechnology 68:2159-2169.

LambacherN.J., Bruel A.L., VanDam T.J.P.,Szymanska K.,SlaatsG.G., Kuhns S.,McManus G.J., Kennedy J.E.,GaffK., Wu K.M.,etal. (2016) TMEM107recruits ciliopathy proteins to subdomainsofthe ciliary transitionzoneand causesJoubert syndrome. Nat. Cell Biol. 18:122- 131.

HaywoodN.J., WolnyM., Rogers B.,Trinh C.H., Yu S.P.,Edwards T.A. &PeckhamM.(2016) Hypertrophiccardiomyopathy mutations in thecalponin-homologydomainofACTN2 affect actin bindingand cardiomyocyte Z-discincorporation. Biochem.J.473:2485-2493.

BaboolalT.G.,Mashanov G.I.,NenashevaT.A., PeckhamM.&Molloy J.E. (2016) A combination of diffusion andactivetranslocation localizesmyosin10tothe filopodial tip. J. Biol.Chem. 291:22373-22385.

Funding Ourwork wasfundedbyBBSRC, MRCand theBritish HeartFoundation.

Collaborators External: G. Mashanov andJ.Molloy(TheCrick Institute, London).

57 main Insights into the foldingmechanisms of periplasmicchaperones

BobSchiffrin,Julia Humes, AntonCalabrese,PaulDevine, Sarah Harris,Alison Ashcroft, DavidBrockwell andSheenaRadford

Introduction Chaperones are alarge,diverse class of proteinsessentialfor maintaining proteostasis. Most proteins in thecellrequire chaperonesfor correct folding, andmost chaperonesinteract with an arrayofclients. Duringtimes ofcellularstress chaperonesare importantinassisting protein foldingand preventing aberrant interactionswhich could leadtoaggregation.Bycontrast with thewell-studied chaperonesystems whichrequire ATPand otherco-chaperones to promote folding(suchasthe chaperoninsand Hsp70s) themechanisms by whichATP-independent chaperones function remain unclear,despite theirfundamental biological importance.Here we have studiedtwo ATP-independentchaperonesfound in theperiplasm of gram-negative bacteria:Skp andSpy.Skp is a16kDa homotrimer, whichselectivelybinds E-barrelouter membrane proteins (OMPs) within ahydrophobiccavity, facilitatingtheir transport across the periplasm before foldinginto theouter membrane(OM). Spyis16kDa homodimerwitha cradle-likestructure,which is dramatically upregulated to concentrationsof~2mMunder stress conditions. Keyquestionsinclude: (1)how do substratesbindchaperones?(2) what are theconformations of chaperones andsubstrates during binding?and (3)and howare substrates released?Our work aims to gain insightintothe mechanisms of ATP-independent chaperone actionusing abroad rangeofin vitro biochemical andbiophysical techniques,as well as computer simulation andmodelling.

Results Skpinteracts with a>30 OMPs in vivo includingmanyOMPswhich appeartobetoo largeto be accomodated within theSkp hydrophobiccavity, yetall models suggesteda1:1 Skp:OMP stoichiometry. To investigatehow Skpisabletoaccommodate itslargersubstratesweused kineticfoldingassays,nativemass spectrometryand moleculardynamics(MD)simulations to examinethe interactions of SkpwitharangeofOMPs of differentsize. Kinetic assaysshowed that greaterSkp:OMP molarratiosare required to preventthe foldingof16-stranded OMPs comparedwiththose containing8E-strandsintheir native state. Consistentwiththese results ElectrosprayIonisationMass Spectrometry (ESI-MS) combinedwithIon MobilitySpectrometry (IMS) revealedthatSkp formsmultivalent 2:1 Skp:OMPcomplexes withlarger OMPs,and that bindingofOMPsofincreasing size requires dynamicexpansion of theSkp hydrophobic cavity. We furher usedmodelling andMDsimulationstoprovidethe firstglimpsesinto possible structuresofthese multivalentcomplexes. (Figure 1).

Figure 1: modelsofSkp:OMP complexes.Skp in a1:1 complex with (A) an 8-strandedOMP,withSkp coordinates from its crystalstructure, and (B) a16-stranded OMPinvolving expansion of theSkp hydrophobic cavity. (C) and (D) Possible architecturesof2:1 Skp:OMP complexes involvinga16-stranded OMP. Allmodelsare consistent with datafromIMS-MSexperiments.

www.astbury.leeds.ac.uk 58 main To studythe Spy chaperone thesmall, four-helix bundleproteinIm7 waschosenasthe client protein.ITC experimentsrevealed that SpyisabletobindnativefoldedIm7,inadditionto mutants whichare either partially or fully unfolded.Weusedstopped-flowfluorescence kinetic experimentsinwhichfoldedorunfoldedwild-type Im7(in 8Murea), or destabilised Im7 mutants,were rapidlydilutedinthe presence of increasing concentrations of Spy. Thedata showedthatSpy rapid associates with client proteins to prevent aggregation. Remarkably, globally fittingofall fluorescence data to differentkinetic models (Figure2)revealed that Im7 is able to fold andunfoldwhile in complex with Spy, providing thefirst evidence that substrates can fold whileremaining boundtoachaperone.

Figure 2: kineticmechanism forthe folding of Im7inthe presence or absence of theATP-independent chaperoneSpy.Im7 is shownasa multicolored proteinthat is helical in boththe folding intermediate (I)and in thenativestate (N) andlacks persistentsecondarystructure in the unfolded state(U).Spy is shownasa light blue/dark bluecradle-shaped homodimer.Spy bindingand release areshown as vertical arrows.

We are nowusing additional experimental techniques to mapclient-chaperone interactions in more detail,aswellasexpanding therange of chaperones andclientproteinsstudied. Our aimistogainfurther insightinto themolecularmechanisms of periplasmicfoldingfactors, includingthe chaperone-mediated delivery of OMP substratestothe E-barrelAssembly Machinery(BAM) forfoldinginthe OM.

Publications SchiffrinB., CalabreseA.N., Devine P.W.A.,Harris S.A., Ashcroft A.E.,Brockwell D.J. & RadfordS.E.(2016)Skp is amultivalent chaperone of outer-membrane proteins. Nat. Struct. Mol. Biol. 23:786-793.

StullF., Koldewey P.,HumesJ.R., RadfordS.E.&BardwellJ.C.A.(2016)Substrate protein foldswhile it is bound to theATP-independentchaperone Spy. Nat. Struct.Mol.Biol. 23:53- 58.

Funding This work wasfundedbythe BBSRCand theNIH.

Collaborators External: J. Bardwell (NIH, USA).

59 main The molecular details of amyloid fibril-glycosaminoglycan binding

Katie Stewart andSheenaRadford

Introduction Amyloidplaques contain an assortment of moleculesthatco-localizegenerically withamyloid fibrils,including metalions, nucleicacids,and glycosaminoglycans(GAGs), linear negatively chargedcarbohydrates.Previouswork fromour laboratory indicatedthatthe GAGheparin LQWHUDFWVGLIIHUHQWO\ ZLWK $ȕ ILEULOVZLWKGLIIHUHQW VWUXFWXUHV ELQGLQJVWURQJO\WRDWKUHH-fold symmetric morphology (Kd=34µM±12 µM), andarguing that structure, in addition to sequence,plays aroleinheparin:fibril binding.Usingsolid-state NMR(SSNMR)and an in vitro bindingassay,wehaverecentlymapped themoleculardetails of bindingfromthe SHUVSHFWLYHRIERWKWKH $ȕ ILEULO DQGWKH *$* 2XUUHVXOts reveal atight and specific interaction, with theheparin chain fullyconstrained in contact with thefibril.Binding involves aclusterofpositivecharges on thefibril aligningwithspecificnegativelycharged sulfates on lowmolecularweightheparin (LMWH).The tightcoordinationobserved between3Qamyloid fibrils andLMWHilluminates howdifferences in fibril or GAGpolymorphs maycontribute to WKHGLYHUVLW\ LQ PDQLIHVWDWLRQDQG SURJUHVVLRQRI$O]KHLPHU¶V GLVHDVH

Detection of octasaccharide bindLQJWR$ȕ 4 ILEULOV DORQJWKH ILEULOD[LV $ȕ ILEULOVZLWKD4 PRUSKRORJ\ZHUH SUHSDUHGE\VHHGHGHORQJDWLRQDQG WKHLUPRUSKRORJ\ wasverified bySSNMR.Tomap theLMWHbindingsite, a 13C-labelledoctasaccharide construct(ashorterproxy of LMWH with similarbindingto3Qfibrils) wasaddedto15N-labelled 3Q fibrils (Figure1A), allowing interaction crosspeakstobemapped by SSNMR upon fibril:octasaccharide binding. Thespectra indicatedthatthe GAGwas fullyconstrained in the presence of 3Q fibrils,suggestingthatthe LMWH and3Qfibril share multiple contactsalong thefibrilaxis(Figure 1A and1B).Chemical shift differencesinthe presenceand absence of theoctasaccharide suggested in particular that oneormore histidine residues on the3Qfibril are involved in binding.

Figure 1: heparin octasaccharide- ELQGVWR$ȕ 4 ILEULOV (A) Proposed bindingbetween 3Q fibrils (green, from PDB file 2LMQ) and LMWH (blue, with one binding site shown),and adetailedviewofthe 13C-labelled octasacchride shown above. (B) 13CCP-MASspectrumof theoctasaccharide bound to 3Q fibrilsat4°C; actual (black) and simulated (red, upper)and 13C-13C spectrumwithDARRmixingtimeof 20 ms (lower).

Theroleofamino acid sidechainsonfibril:heparin binding. To investigatethe role of positivelycharged residues on the3Qfibrilasheparin bindingsites, VLQJOH VXEVWLWXWLRQYDULDQWVRI$ȕ ZHUH GHYHORSHGWKURXJKRXWWKH SURWHLQ %LQGLQJRI4- seededvariants wasmeasuredusing an in vitro assayinwhich theLMWHbound to variant $ȕ ILEULOVZDV SHOOHWHG E\ FHQWULIXJDWLRQDQG WKHVDFFKDULGHFRQWHQW LQ WKHVXSHUQDWDQW ZDV quantified by enzymatic digestionwithheparinaseIIfollowedbyUVspectroscopy. Thelargest ǻǻ*ƒRIEindingoccurredinresidues locatedatthe N-WHUPLQXV DQG FHQWUDOORRSRI$ȕ,Q the3Qfibrilstructure,these regionsare spatially adjacent,forming acluster of positively charged residues whichappearvital forLMWHbinding(Figure 2A).

www.astbury.leeds.ac.uk 60 main Theroleofheparin substituents on fibril:heparinbinding. We assessedthe role of theheparinmolecule in bindingamyloid fibrils by removingsulfate groups fromthe heparinchain systematically andtesting bindingtoWT3Qfibrils by the in vitro assay.The resultsindicated that removalofthe 2-O-sulfatedid notreduce binding, but removal of the6-O-sulfate eliminated heparinbinding (Figure2B).The NS sulfate also reducedbinding, butthe effectsvaried by thesubstitution, indicating that thespatial arrangement of negative charges on heparinisimportant.These results were further confimed by seededaggregation kinetics (using thioflavin Tfluoresence), in whichthe 3Q-VHHGHG$ȕ aggregationratechanged in thepresence of LMWH butnot in thepresenceofheparin without theNSand 6-Osulfates YHUVXV $ȕ VHHGHG ZLWKRXWKHSDULQ

Figure 2: substitutions to the 3Q fibrilorheparin affect binding. (A) 7KH$ȕ 4 ILEULO structurewithsubstituted residues colouredfromsmall (blue) to large (red) changesin ǻǻ*ƒELQGLQJ (B) Heparin disaccharidewithsulfates labelled andcomparisonof ǻǻ*ƒELQGLQJ of modified heparinsto3Qfibrils. Asterisksdenoteheparins whichshowed little to no binding.

Tightand specific coordinationare keytofibril:heparin interaction. 2XUUHVXOWVVXJJHVW WKDW WKHLQWHUDFWLRQEHWZHHQ 4 ILEULOV RI $ȕ DQGKHSDULQ DUHGULYHQE\ complementary charges packed into specificarrangements andare notsimplythe result of electrostatic patterning,asisthe case in other accessory moleculessuchasnucleic acids. Instead, thetight andspecificbindingweobserveisthe result of aprecise andcompatible alignment of charges.These resultssuggest that theprevalence of plaque-associated GAGs PD\EHGLFWDWHG E\ DSDWLHQW¶V ILEULO morphology(ies), andmay playarole in theprogression DQGVHYHULW\ RI $O]KHLPHU¶V GLVHDVH

Publications StewartK.L., Hughes E.,Yates E.A.,Akien G.R., HuangT.Y., Lima M.A.,RuddT.R., Guerrini M.,HungS.C., RadfordS.E.,etal. (2016)Atomicdetails of theinteractionsof glycosaminoglycanswithamyloid-betafibrils. J. Am.Chem. Soc. 138:8328-8331.

Funding This work wasfunded by theBBSRC.

Collaborators External: D. Middleton (LancasterUniversity),E.Yates (University of Liverpool), S.-C.Hung (GenomicsResearch Center,Academia Sinica, Taiwan).

61 main /DWHUDO RSHQLQJ LQ WKH LQWDFW ȕ-barrel assembly machinery captured by cryo-EM

Anna Higgins, MatthewIadanza,Bob Schiffrin, AntonioCalabrese,Alison Ashcroft, David Brockwell, Sheena Radford and Neil Ranson

Introduction Outermembrane proteins (OMPs)mediate thesurvival andpathogenicityofGram negative bacteria.The biogenesisofthese proteinsiscomplex,however, astheyare synthesised in thecytoplasm,are transportedbychaperonesacrossthe periplasm,and must then be LQVHUWHG DQGIROGHGFRUUHFWO\ LQWR WKHEDFWHULDORXWHU PHPEUDQH LQ WKHDEVHQFH RI $73 7KHȕ- barrelassembly machinery(BAM)complexofE.coli is a~203 kDaassemblyoffiveproteins (BamA-E)thatenables themembraneinsertion andfoldingofsubstrate OMPs on a physiological timescale.Despitethe availabilityofcrystal structuresfor each of thefiveBAM subunits, various subcomplexes, OMPsubstrates, andmostrecently thefull E.coli BAM complex,the mechanismofthisvital protein assemblyisunknown.Anunderstanding of how BAM-mediated OMPfoldingand insertionoccurs will provide insightinto OMPbiogenesisin E. coli,and potentiallyofhomologous proteins in theouter membranes of mitochondria and chloroplasts. Also, becauseBAM is surface-located, essential,and conserved,itisan attractivepotential target forthe developmentofnovel antibacterials.Herewehaveuseda variety of structuraland biochemicaltools to probethe nature of BAM-assistedOMP folding.

Results 7KHȕ-barrelofBamA is knowntoexist in twoconformations: in thefirst it is sealedonits H[WUDFHOOXODUIDFH DQGRSHQWRWKH SHULSODVPDQG LQ WKHVHFRQG WKHUHLVDVHSDUDWLRQRIWKH ȕ DQGȕ ȕ-VWUDQGV RI WKHȕ-barrel, creatingastructure“lateral open” to theouter membrane andextracellularface. Such conformational changes aresignificantfor several reasons. Firstly as BamA is hypothesised to assist substratefoldingthrough itslateralopening,suchflexibility maybecritical forthe BamA functional cycle. Secondly, thus farlateralopen conformations have only been observed in %$0 FU\VWDOVWUXFWXUHVZKLFK ODFN WKHN'Dȕ-propeller protein BamB, raisingthe possibilitythatchanges in BamA mayrepresent agatingreactiondriven by BamB binding.

Figure 1: threeviews of a4.9Å cryo-EM mapofthe BAMcomplex with afittedpsuedo-atomicmodel, showing thecomplex in the laterally open conformationinthe presenceofBamB.

Utilisingthe BAMcomplex recombinantly expressed andpurifiedfrom E.coli outermembranes in DDMdetergent micelles, we were able to elucidate thefirst cryo-electron microscopy structure of thefullcomplex,ataresolutionof4.9 Å1 (Fig.1). Thestructure reveals distinct conformationalchanges relativetoexistingcrystal structures and, forthe firsttime, reveals interactions betweenBAM subunitsand thedetergent micelle, suggestingmodes of communicationbetween BAMand thelipid bilayer. Additionally, thestructure reveals theintact BAMcomplexinwhich BamA is in alaterally-openconformation,withseparationbetween the ILUVW ȕ DQG ODVW ȕ VWUDQGVRIWKH EDUUHOLQWKH SUHVHQFH RI %DP%-E.The suggestionthat BamA lateralopeningisimportant foractivityinsubstrate foldinghas been supported by in vivo data showing that disulfide cross-linking of thestrandsislethalinE.coli.Toexamine

www.astbury.leeds.ac.uk 62 main whetherlateralgatinginBamA is required forBAM-mediated OMPfolding in vitro,wecreated thesameBamAmutantsdesignedtocross-link E1-E16 andexamined theability of BAM containingmutantBamA to fold thesubstrate OmpT (Fig.2a).

OmpT foldingwas assayed using aself-quenching fluorogenic reporter peptidewhich is cleavedbythe correctlyfolded/membraneinserted endoproteaseOmpT. In E.coli polarlipid liposomescorrect foldingofOmpTisknown to requirethe presence of active BAMand the OMP-chaperone,SurA.While proteoliposomes reconstituted with thewild-type BAM, or containingaCys-freeBamA mutantshowhighlevelsofactivityinfoldingOmpT, BAM proteoliposomes that incorporatedisulfide cross-linkedBamAshowsignificantly loweractivity (Fig.2b,c),indicatingthatthe disulfide cross-OLQNDFURVV WKHȕ-barrelimpairs, butdoesnot prevent, BAM-mediated foldingofOmpT in vitro.Consistentwiththisnotion, additionof reducing agent rescuesBAM activitytonear wild-type levels.

Figure 2: importanceofBamAlateral gating in BAM complexfunction. (A) Thepositionofthe natural cysteines of BamA (replacedwithSer)are indicatedwitharrows, and thosethatwereintroducedtoformadisulfide cross- link areindicated by yellow spheres. (B) Example traces of fluorescencedue to OmpT foldingcomparing BAM proteoliposomes containingdisulfide mutant (purple) BamA,wild-type (blue) andCys-free(green)BamAin reduced andoxidisedconditions. (C) Barchart showing average half-timefor each reaction.

Ourfunctionalassays providethe first in vitro evidence of theimportance of BamA lateral gating in BAMfunction, demonstratingthatlateralgatingofBamA diminishes, butdoesnot eradicate,the abilityofBAM to assist OmpT folding. Additionallythe cryo-EM structureshows that BamA lateralopening occurs independently of BamB binding.Current work is focusing on using othermembrane mimetics forcryo-EM structureand otherfunctionalassays to determinethe molecularmechanism(s) of BAM function.

Publications IadanzaM.G., HigginsA.J., SchiffrinB., Calabrese A.N., BrockwellD.J., Ashcroft A.E., RadfordS.E.&Ranson N.A. (2016) Lateralopening in theintact E-barrelassembly machinery captured by cryo-EM. Nat. Commun. 7:12865.

Funding This work wasfunded by TheWellcomeTrust andthe BBSRC.

63 main Coordinate regulation of antimycin andcandicidin biosynthesis

Thomas McLean andRyanSeipke

Introduction Streptomyces species produce an incrediblearray of high-value specialty chemicalsand medicinaltherapeutics. Asinglespecies typicallyharbours ~30 biosynthetic pathways,but only afew them are expressed in thelaboratory;thus, poorunderstanding of hownatural- product biosynthesisisregulated is amajor bottleneck in drugdiscovery. Antimycins are potent antifungal compounds made by Streptomyces bacteria that arealsoselectiveinhibitors of theBcl-2/Bcl-xL-family of anti-apoptoticproteinswhich areoften over-producedindrug- resistantcancers. Antimycins areproducedbyahybridnon-ribosomal peptidesynthetase / polyketidesynthase biosyntheticpathway whichweidentifiedand characterised in Streptomycesalbus.The biosynthetic gene clusterconsistsof15genes organised into three operons: antBA, antCDE, antFG and antHIJKLMNO. Thegenecluster harbours asingle cluster-situatedregulator,anorphanalternative RNA polymerase sigma factorencoded by antA ıAntA  ıAntA regulatesthe expression of antFG and antHIJKLMNO, whichdirect the biosynthesis andactivationofthe 3-formamidosalicylate precursor, butnot antBA or antCDE. Here we showthatFscRIco-ordinately regulates productionofbothcandicidinand antimycin.

Results AseriesofbioinformaticsanalysessuggestedthatFscRImay regulate antBA and antCDE. To test this hypothesis,wedeleted the fscRI gene in Streptomycesalbus usingCRISPR/Cas9 JHQRPH HGLWLQJDQG WHVWHG WKHUHVXOWLQJPXWDQW ¨fscRI)for theproductionofantimycins. As SUHGLFWHGWKH ¨fscRI strain could no longer produce antimycins, aphenotype whichwewere able to rescuebyectopically expressingfscRI from aconstitutivepromoter.Wethen performedchromatin immunoprecipitation sequencing using a3xFLAG-tagged variant of FscRI. Theresultsrevealedthat3xFLAG-FscRIbinds to the antBA and antCDE promoter regions in vivo.Our studyprovides direct in vivo evidence of thecross-regulationofdisparate biosyntheticgene clusters specifying unrelatednatural products andexpands the paradigmatic understanding of theregulationofsecondary metabolism.

Figure 1: modelfor theregulationofantimycin biosynthesis: (top) therelative locationsofthe antimycinand candicidingene clustersinthe S. albus chromosome. (bottom) FscRIactivates transcription of antBA and antCDE,whichresults in production of thecoreAntC/AntD NRPS/PKS megasynthase and productionofthe discreteacyltransferaseAntBand AntA, which in turn activatestranscriptionofthe ketoreductase antM and nine genes(antFGHIJKLNO)required forthe biosynthesis and activation of the3-formamidosalicylate precursorutilised by AntC. AntA does notpossess acognate anti-sigmafactor and instead appearstobeinactivated by the ClpXPprotease.

Publications McLean T.C.,HoskissonP.A.&Seipke R.F. (2016) Coordinateregulationofantimycin and candicidinbiosynthesis. mSphere 1:e00305.

Funding This work wasfunded by theBBSRC.

Collaborators External: P. Hoskisson(University of Strathclyde).

www.astbury.leeds.ac.uk 64 main Studyofsensory neuronsofC.elegans usingamicrofluidic device

JinyangChung andJung-ukShim

Introduction Caenorhabditiselegans (C.elegans)isaroundworm in thephylumofnematode.Ithas a simple buthighlydeveloped neuron system.Especially,12pairs of anatomically symmetrical sensory neuronsare located left andright side in thehead. Among them,ASH neuron is knownasanociceptortosense theosmotic,chemicaland mechanical stimuli, whichmay likelydamagethe organism.And,ASE neuron is afunctionally asymmetricchemosensory neurons e.g. sensing NaCl;ASEL responds to theincreaseofNaClconcentration andASER to thedecrease of it.However,itischallengingtoinvestigate both neuronsatonceonasame focalplane with atypical microscope slidebecause theworm swam or/andcrawledwithits side orientatedout-of-plane. Theaim of this studyistodevelop an approach, whichwould enable us to immobilize C.elegans, controlmicroenvironmentand investigateits sensory neurons in dorso-ventralaspect.Here, we report that amicrofluidicdevicehavebeen developedtotrapthe worm,treat with thestimulusand imagethemfromthe side-view (dorso- ventralview).Ithas enabledustoobserveapairofthe neuronsinthe same focalplane.A worm wasgenetically modifiedtoexpress calcium indicator protein(GCaMP) inits ASH/ASE neurons, immobilizedinthe device andthe stimulus (NaCl) wastreated to theamphidofthe worm.

Results Developmentofamicrofluidic device Amicrofluidicdevicehas been developedbypolishingthe side of thechannel in orderto observeapair of neuronsinthe same focalplane. Thestimuluschannel is asymmetrically placed in thedevicetosecure theworking distance of theobjective lens.The entire device wasbuilt with PDMS;moulded PDMS block wascovalentlybound on other PDMS plate via theoxygenplasmatreatment andthe device wascut alongthe trapchannel.The thicknessof thePDMSfromthe surface of thesidetothe channelwas approximately2mm,which was withinthe workingdistanceofthe microscope lens. Response of ASHneuronstothe stimulus throughthe side-view TheASH-worm wasinjectedand immobilized in thetrapinthe device by applicationofthe external pressure.The device enablesustoincubate theimmobilized worm formorethan30 minutes. Theamphid of theworm exposedtobuffer/stimulus through thetrap-orifice in the flowingchannel.The stimulus (500 mM NaCl) is treatedtothe worm using theflowcontrol system. Cell bodies of theneuronand ASHresponse dendriteselongated to 800 Figure 1: thefluorescenceof theamphidare Left 600 GCaMPexpressedonthe

a.u.) Right fluorescently visible ASH neuron is measuredwith y( andthe intensity scence 400 theapplication of thestimulus re

nsit (500 mM NaCl). Three increases when the 200 te repeated pulses (10seconds Fluo

amphidtouched the in 0 on and10seconds off) of stimulus stream, 100150 200250 stimuluswas applied. whichindicates Ca ion Time (s) intakeand the depolarization of theneuron.

Collaborators University of Leeds: C. Brittin, N. Cohen.

65 main Simvastatin and fluvastatin interact withhuman gapjunction gamma-3 protein

Paul Taylor

Introduction Findingpleiomorphic targetsfor drugsallowsnew indications or warningsfor treatmenttobe identified. As test of concept, we appliedanewchemical genomics approach to uncover additional targetsfor thewidelyprescribed lipid-loweringpro-drugsimvastatin.

Results We usedmRNA extracted frominternal mammary arteryfrompatientsundergoingcoronary artery surgery to prepare aviral cardiovascular protein library, using T7 bacteriophage.Wethenstudied interactions of clones of thebacteriophage, each expressing adifferent cardiovascularpolypeptide, withsurface-bound simvastatin in 96-wellplates. To maximise likelihoodofidentifyingmeaningful interactions betweensimvastatin andvascular peptides, we used avalidated photo-immobilisation method to applyaseriesofdifferent chemical linkers to bind simvastatinsoastopresent multiple orientations of itsconstituentcomponents to potentialtargets.Three rounds of biopanning Figure 1: discovered N-terminal residues identifiedconsistentinteractionwiththe clone 1–25 (orange). expressing part of thegene GJC3,which maps to Homo sapiens chromosome 7, andcodes forgap junction gamma-3protein,alsoknown as connexin30.2. Furtheranalysisindicatedthe binding site to be forthe N-terminaldomainputatively‘regulating’connexinhemi-channel andgap junction pores. Usingimmunohistochemistrywefound connexin30.2tobepresentinsamples of artery similartothose usedtoprepare thebacteriophagelibrary. Surface plasmon resonance revealedthata25 aminoacidsyntheticpeptide representing theuncovered N- terminus didnot interact with simvastatin lactone, butdid bind to thehydrolysed,HMG CoA inhibitor, simvastatin acid. This interactionwas alsoseenfor fluvastatin. Thehemichannel blockerscarbenoxoloneand flufenamic acidalsointeracted strongly withthe same peptide providing novelinsightinto their potentialmechanismofinhibition. Thesefindings raisekey questionsabout thefunctionalsignificanceofGJC3 transcriptsinthe vasculature andother tissues, andthisconnexin’sroleintherapeutic andadverse effectsofstatinsinarange of disease states.

Publications Marsh A.,Casey-Green K.,Probert F.,Withall D., Mitchell D.A.,Dilly S.J., JamesS., Dimitri W.,Ladwa S.R.,TaylorP.C., et al.(2016)Simvastatin sodiumsaltand fluvastatin interactwith humangap junction gamma-3 protein. PLoS One 11:e0148266.

Funding This work wasfunded by EPSRC,Tangent ReprofilingLtd.&AdvantageWestMidlands.

Collaborators External: A. Marsh, K. Casey-Green, F. Probert, D. Withall, D.A. Mitchell, S.J.Dilly,S.James, W. Dimitri, S.R. Ladwa, D.R.J. Singer(University of Warwick).

www.astbury.leeds.ac.uk 66 main Positiveallosteric modulation of TRPV1 usingAffimerreagents

RobBedford,MikeMcPherson andDarrenTomlinson

Introduction TRPV1 is amajor target forthe management of chronicpain andspecific modulators are highly soughtfor theiranalgesicpotential.Despitepromising resultsusing TRPV1 inhibitors forthe management of chronic pain,anumber of severe side effectsresulted in their withdrawal from clinical trials. Conversely,TRPV1 activationhas been exploredasan alternativemechanismofpainmanagement, yetanumber of side effectsstill occur owingto targetingofnon-pathological receptors.Anovelapproach targetingonly activated receptors selectively desensitises TRPV1 at sitesofchronic pain only;thisistermedpositiveallosteric modulation.Our work demonstratesthe selection of Affimerreagents able to positively modulate TRPV1 by bindingofthe outerporedomain.

Results Affimers area91 amino acidscaffold with tworandomised loop regionsfor molecular recognitionthatare selected using phagedisplay.Initially,apeptidesequence takenfromthe TRPV1 outerporedomainwas screenedusing phagedisplay.Phage ELISAsubsequently confirmedAffimerbinding.TRPV1 specificcloneswere sequencedand,interestingly,results revealed homology of Affimerloops with theTRPV1-interactingloopofDkTx, atoxin that activates thereceptorbybindingthe peptideregionscreened. Affimers were subsequently expressedand theirabilitytobindtoTRPV1 inmammaliancells assessed by affinity fluorescence.Anumberofthe Affimers showedspecificmembrane staining (Figure 1).

Figure 1: immune-cytochemical staining of TRPV1 using Affimers. TRPV1 receptor was overexpressedonamammalian cell lineand incubated with Affimer reagents.Results revealed membrane staining of cellsover-expressing TRPV1 compared to anegativecontrol cell linesuggestingAffimerswere able to identifyfull-lengthTRPV1.

Next we determined theability of Affimers to modulate TRPV1.Despitehomologytothe toxinthatactivates TRPV1, theAffimershad no direct effect.However, Affimers demonstrated positive allosteric modulationofTRPV1 when activated by capsaicin(Figure 2). Takentogether, theseresultsdemonstrate theidentificationand modulationofaclinically relevant ionchannel,TRPV1,using Affimerreagents.

67 main

100 n=3 **** 80 Figure 2: positive allostericmodulation of *** TRPV1 usingAffimers. TRPV1 was 60 * * * overexpressedonmammaliancells and RFU * 2+

' incubated with theCa dye, Fluo4-AM.Using a

% 40 Flexstationmultiwall platereader,cells were stimulatedwith0.2Mcapsaicinand thechange 20 in fluorescencemeasured. Results demonstratedthatpresenceofAffimerscaused 0 an increase in capsaicin-inducedactivationof

r1 r2 r3 r4 r5 r6 r7 r8 TRPV1, compared to cells exposed to control nly r11 r10 r12 r13 r14 mer Affimer. me me me me me me me me ro me me me me me fi fi fi fi fi fi fi fi Affi fe Af Af Af Af Af Af Af Af ol Affi Affi Affi Affi Affi Buf tr Con

Future work will nowfocusonunderstanding theinteractionbetween Affimerreagentsand TRPV1 at amolecularlevel. Insights provided by this mechanismofTRPV1 modulationmay aidfuture drug discovery forthe treatment of chronicpain.

Funding Neusentis,Pfizerand theUniversity of Leedsfundedthiswork.

Collaborators University of Leeds: J. Lippiat.

www.astbury.leeds.ac.uk 68 main Regulationofmusclecontraction by titinand myosin bindingproteinC

CharlotteScarff, AlbaFuentes Balaguer, MehrnazMontazeri, Larissa Tskhovrebova and John Trinick

Introduction Titinisthe largestknown protein (chain weight ~3.5 MDa) andthe thirdmostabundantprotein of muscle,aftermyosinand actin.Individual titinmoleculesspanbetween theZ-and M-lines andinthe A-band are attached to thesurface of themyosinfilamentshaft.Untilrecently vertebrate striatedmuscle contractionwas thoughttoberegulated solelybyblockingof myosinaccess to actin in thin filamentsbytroponinand tropomyosin. Recently,however,there hasbeenmountingevidence that titinisinvolvedinactivatingmyosin. Thedataindicate that titinactsonmyosinbindingproteinC(mybp-C, alsoknownasC-protein), whichinturn acts on forceproducing myosincross-bridges, acceleratingtheir cyclingrateonactin. PhosphorylationofC-protein also speeds therate of cross-bridge cycling.

Results We expressed whole human C-protein (150 MDa) in insect cells andour collaborator, Donald Winkelmann, suppliedexpressedhuman heavymeromyosin(HMM),which is theN-terminal half of myosinthatforms cross-bridgeswithactin.Wehavecarriedout electron microscopy on theC-protein andonthe complex it formswith HMM. Negativelystained imagesofC- proteinalone showthe individual immunoglobulin (C2) and fibronectin (type3)domains from whichthe molecule is constructed. TheimagesshowaV-shaped moleculeabout40nmlong and 3nmwide. Thereisoften akinkinone of thearms, whichprobably is theN-terminalend of themolecule.Weare also usingelectronmicroscopytoimage theC-protein/HMM complex, usingbothnegativestainingand cryo-EMoffrozenhydrated specimens.Apreliminary 3D modelofthe complexhas been produced fromthe staindata.

Figure 1: arrangement of titinand C-proteininvertebrate striated muscle sarcomeres.The titinmolecule spansbetween theM-and Z-lines.C-protein (verticalbars) binds to bothtitin andmyosinatseveral positions spaced 43 nm in theC-zone of the A-band.

Funding Funded by theBritish Heart Foundation andthe Leverhulme Trust.

Collaborators External: D. Winkelmann (Rutgers University).

69 main Co-operativemechanism of protein translocation through Sec machinery

Peter Oatley,Tomas Fessl, Stephen Baldwin, Sheena Radford and Roman Tuma

Introduction Proteintransport across thebacterial innermembraneoccurs primarily viathe Sectranslocon. Thetransloconcore,SecY,comprises10trans-membrane helices (TMs)ina‘clamshell’ arrangementforming acentralchannel throughthe membrane.Alateralgate(LG)islocated betweenTMs 2, 3and 7(Fig. 1A)and provides an exit pathwayfor thesignal sequence andfor membrane protein insertion into themembrane. In bacteria, secretionisgenerally post-translational andsubstratesare recognised by the cytosolic ATPase SecA which subsequentlyassociateswithSecYEG. TheATPase acts as amolecularmotor anddrives thesubstrate through the protein-channel using energy from ATP bindingand hydrolysis togetherwith proton motiveforce.WithinSecA,two RecA-likenucleotidebinding domains (NBD1and NBD2) form thenucleotide- bindingsite(NBS), connected to a‘two- helixfinger’ (2HF), andapre-protein cross-linking domain(PPXD,Fig.1A). Upon SecA bindingtoSecYEG,the 2HF penetrates into theproteinchannel of SecY andthe PPXD formsa‘clamp’ for thesecretory substrate.Several structuresofSec-translocons have been determined in thepresenceofvarious substrates, in both open andclosed conformations.However, themechanism throughwhich ATPhydrolysisiscoupled Figure 1: (A) structureofSecYEG:SecA complex and to directional movementofsubstrate is schematic depictionofthe translocon(left). (B) unclear. Here,wecombine all-atom Schematics of proteoliposome immobilisation forsingle moleculardynamics(MD)simulations molecule imaging. (C-H) FRET efficiency histogramsfor different states: (C) SecYEG only, (D) with single molecule FRET and SecYEG:SecA:AMP-PNP, (E) SecYEG:SecA:ADP, (F) biochemicalassays in order to address SecYEG:SecA:ATP, (G) SecYEG:SecA:ATP:pOA, (H) allosteric mechanismofcoupling SecYEG:SecA:ATP:pOA with excess of SecA. betweenATP hydrolysisatSecA and proteintranslocation throughSecY.

Results MD simulationwere initiated fromatransition statestructure (SecA:ADP:AlFx)but with SecA nucleotide bindingpocket occupiedwitheitherADP or ATP(AMP-PNP). TheADP-bound structure convergedinto state with LG closed whileATP bindingbiasedthe SecY LG towards an open state.Overall, nucleotideexchangeatSecAelicitednanometer motionswithinthe distal LG of SecY.Inorder to monitor thesemotions we have positionedanAlexa FluorFRET pair (AF488 andAF594)acrossthe LG andreconstituted SecYEG into unilamellarlipid vesicles whichwere then immobilised on aglass slidecover slip forimaging with total internal reflection microscopy(Fig. 1B). Singlemolecule fluorescencetraceswere collected for differentstates,converted into FRET efficienciesand collated into histograms (Fig.1C-H).In

www.astbury.leeds.ac.uk 70 main

Figure 2: aBrownian motormodel fortranslocation of unfolded polypeptide chain through Sectranslocon. AfterinitiationSecA staysinthe ADP state(i),forcing LG to theclosedconformation throughwhichonly smallside chains candiffuse. Upon encounteringacharged or abulky side chain(ii) 2HFtriggers nucleotideexchangeatSecAfor ATP(iii) whichinturnopens LG to allow diffusion of thebulky residues (iv).UponATP hydrolysis LG closes (v)blocking there-entry of thebulky side chain from the periplasmicside(vi). Aftermultiplecyclesthe translocation processcompletes by acleavage of the signalpeptide.

theapo-SecYEG stateLGremainedclosedasmanifestedbyhighFRETefficiency(closely spaced dyes).However,uponadditionofSecA andAMP-PNP LG populatedopen conformationwhile in thepresence of ADP it wasmostly closed. This reproducedthe results from MD simulations andconfirmed existenceoflong-rangeallosteric control of LG opening by nucleotide state of SecA.Furthermore, in thepresence of ATPadditional, partlyopenstate wasdetected (Fig.1F) whileduringtranslocation of pro-OmpA substrateLGco-existedinall three states,indicatingnucleotide-dependent changes in thechannel aperture during translocation.Crosslinking of theLGinthe closedstate promoted ADPbindingwhile crosslinking of ashort oligopeptide containingbulkysidechains(atranslocating chain mimetic)to2HF promotedADP release, whichconstitutesthe rate limitingstep. This indicates that theLGstate andthe two-helix finger environment are both communicated back to the SecA ATPase site.Hence,there exists atwo-way communication betweenthe SecY-channel andthe nucleotide bindingsiteofSecA:ATP bindingcausesopeningofthe channel,while bulkysubstratesatthe channelentranceregulatenucleotideexchange. Thedatasuggest a novelmechanism forbiological motors that transportproteinsand nucleicacids, wherebyATP turnover provides force by biasing thedirection of substratediffusion (Fig.2).

Publications AllenW.J., Corey R.A.,OatleyP., SessionsR.B., BaldwinS.A., RadfordS.E., Tuma R. & CollinsonI.(2016)Two-way communicationbetween SecY andSecA suggests aBrownian ratchetmechanismfor proteintranslocation. eLife 5: e15598.

Funding This work wasfundedbythe BBSRC.

Collaborators External: W. Allen, R. Corey, R. Sessionsand I. Collinson(University of Bristol).

71 main The roleofstructured RNA in early virus replication events

Andrew Tuplin

Overview Aphylogenetically conservedRNA structure within theNS5Bcodingregionofhepatitis Cvirus functionsasacis-replicatingelement(CRE).Integrity of this CRE, designated SL9266,is critical forgenomereplication. SL9266 formsthe core of an extended pseudoknot,designated SL9266/PK,involving long distance RNA-RNA interactions betweenunpairedloops of SL9266 anddistalregions of thegenome. Previous studies demonstrated that SL9266/PK is dynamic, with'open'and 'closed'conformationspredictedtohavedistinct functions during virus replication. Usingacombination of site-directedmutagenesisand locked nucleicacids(LNA) complementary to defineddomains of SL9266and itsinteractingregions,wehaveexplored theinfluence of this structureongenome translationand replication. We demonstrate that LNAs whichblock formation of theclosedconformationinhibit genome translation. Inhibition wasatleast partlyindependentofthe initiationmechanism, whetherdrivenbyhomologous or heterologousinternalribosome entrysites or from acapped message.ProvisionofSL9266/PK in trans relievedtranslational inhibition, andmutationalanalysisimpliedamechanisminwhich theclosedconformationrecruits acellularfactorthatwould otherwisesuppressestranslation. We proposethatSL9266/PK functionsasatemporal switch,modulatingthe mutually incompatible processesoftranslationand replication(Figure 1).

Figure 1: Acartoon interpretation of SL9266/PK conformational rearrangement as part of atranslation/replicationswitch; illustratingthe mechanismand role of negative feedbackinteractionsinthe proposedmodel.The uppercartoonillustratesup- regulation of HCVIRES initiated translation (largered arrow)due to theSL9266/PK closed conformation sequestering acellular IRES inhibitionfactor(IF). Thelower cartoon illustratesthe open conformation, initiated by virally encoded non-structural proteins (5Aand 5B)bindingthe 3’NCR andblocking ‘kissing loop’ formation. Theopen structurefavours interaction between cellular proteinEWSR1 and SL9266, up- regulating RNAreplication (large blue arrow).Asdemonstratedinthe current studythe resultingopenconformationactivelyinhibitsHCV translation(small red arrow),which we speculateisdue to EWRS1displacing -and thus making available -translation inhibitionfactorIF.

Publications KhasnatinovM.A., Tuplin A.,Gritsun D.J.,SlovakM., Kazimirova M.,Lickova M.,Havlikova S.,Klempa B.,LabudaM., GouldE.A.,etal. (2016)Tick-borneencephalitis virusstructural proteins arethe primaryviral determinants of non-viraemic transmissionbetween ticks whereasnon-structuralproteins affect cytotoxicity. PLoS One 11:e0158105.

Stewart H.,Bingham R.J.,White S.J.,Dykeman E.C.,Zothner C.,TuplinA.K., Stockley P.G., TwarockR.&Harris M. (2016) Identificationofnovel RNA secondary structures within the hepatitisCvirusgenomereveals acooperativeinvolvementingenomepackaging. Sci. Rep. 6:22952.

Funding This work wasfunded by aMRC NewInvestigator Research Grant.

Collaborators University of Leeds: M. Harris,A.Zhuravleva,N.Stonehouse andJ.Mankouri. External: A. Kohl (MRC Centre forVirus Research, UK);A.Davidson(University of Bristol, UK) andA.Merits (UniversityofTartu,Estonia).

www.astbury.leeds.ac.uk 72 main Novelantiviral strategies forhuman herpesviruses

Sophie Schumann, Belinda Baquero-Perez,CharlotteRevill, Richard Fosterand Adrian Whitehouse

Introduction Human herpesvirusesare responsiblefor arange of acute andchronic diseases, including several cancers.Kaposi’s sarcoma-associated herpesvirus(KSHV) isthe etiologicagent of Kaposi’ssarcomaand twolymphoproliferative disorders;primary effusion lymphoma and multicentric Castleman'sdisease. Like allherpesviruses, KSHVhas twodistinctforms of infection; latency andlytic replication.While themajority of KSHV-associated tumorigenic cells harbourlatentvirus, lyticgeneexpression occurs to various levels in each KSHV-associated disorder, suggesting that lyticreplicationinhibitionmay providetherapeutic intervention. Currently, drugs in clinical use are inhibitors of herpesvirus DNA polymerasesand arehighly effectiveagainst avariety of herpesviruses. Althoughdrug-resistantstrains areemergingin immunocompromised patients andvarying efficacy hasbeen reported against oncogenic herpesviruses. Consequently,there is an urgent need forthe continued developmentofanti- herpesvirusdrugs,particularly targetingoncogenic herpesviruses.

Figure 1: predictedbindingmodel of hitcompound to theUAP56 ATPbinding pocket.

RNAhelicasescontributetoremodellingofintramolecularRNA-, RNA-proteinand protein- proteininteractions in an ATP-dependentmanner.Both viraland cellularRNA helicaseshave centralrolesinvirus life cycles andhaveemerged as therapeutictargets.Numerousstudies have evaluated thepotential of targetingvirally-encoded RNA helicases. However, to circumventviral resistance,inhibitingcellularRNA helicaseshas also been explored, supportedbyeffortstargeting eIF4Afor thetreatment of cancer andDDX3toinhibit HIV replication, illustratingselectivepharmacological targeting of RNA helicasesispossible. TheKSHVopenreading frame (ORF)57protein,which hasafunctional homologue in each humanherpesvirus, is essentialfor virallytic replication.Itisamultifunctionalprotein involved in allstagesofviral mRNA processing viaan interactionwiththe humantranscription/export (hTREX)complex.hTREX is alarge multiprotein complex involved in Nxf1-mediated cellularbulk mRNA nuclearexport. Notably, ORF57-mediated hTREXrecruitmentproducesaviral ribonucleoproteinparticle(vRNP) essential for KSHVlytic replication. ORF57forms adirect interaction with thecellularexportadapter Aly, however, redundancyinthe cellularmRNA export pathwayalsoallowsORF57 to facilitate vRNP formationvia an interactionwithUIF.Assuch, Figure 2: inhibitionofHSV-1 replicationin disrupting theORF57-hTREXinteractionrequires thepresenceofincreasingamountsofthe hit blockingmultipleprotein-proteininteractions. compound.

73 main Alternatively, thecellularRNA-helicase UAP56functions as an essentialhTREX assembly factor, forminganATP-dependenttrimericcomplex with Alyand CIP29, as well as recruiting furtherhTREX components onto mRNAs.Therefore, we examinedthe potentialofinhibiting UAP56ATPase activity,topreventKSHVvRNP formationand lyticreplication.Insilicohigh- throughputscreening identified smallmoleculescapableofbinding theUAP56ATP-binding pocket (Fig.1). Strikingly,results demonstrate that inhibitingUAP56ATPase activity representsanantiviral target (Fig.2).

Publications Schumann S.,Jackson B.R.,YuleI., WhiteheadS.K., Foster R. andWhitehouseA.(2016) Targetingthe ATP-dependentformation of herpesvirusribonucleoproteinparticleassembly as an antiviralapproach. Nat. Micro. 2:16201.

Schumann S.,Baquero-Perez B. &Whitehouse A. (2016) Interactions betweenKSHVORF57 andthe novelhuman TREX proteins,CHTOP andCIP29. J. Gen. Virol. 97:1904-1910.

Wood J.J.,Boyne J.R., PaulusC., Jackson B.R.,Nevels M.M.,WhitehouseA.&Hughes D.J. (2016) ARID3B:Anovelregulator of theKaposi'ssarcoma-associated herpesviruslytic cycle. J. Virol. 90:9543-9555.

Funding This work wasfunded in partsbyWorldwide Cancer Research, BBSRC andWellcome Trust.

www.astbury.leeds.ac.uk 74 main Designed inhibitors of protein-protein interactions

George Burslem, ClaireGrison,Sarah Hewitt, KatherineHorner, Thomas James,Hannah. Kyle,JenniferMiles, Chris Pask,Silvia Rodriguez-Marin,Philip Rowell, AlexBreeze, Thomas. Edwards, Adam.Nelson,StuartWarriner, Andrew Wilson

Introduction This report summarisesour group’sefforts to developinhibitors of protein-proteininteractions (PPIs) whereweemphasize developmentofconstrained peptides, novelproteomimetics based on aromaticoligoamides andsurface mimetics basedonco-ordination complexes.

Hydrocarbonconstrained peptides –understanding preorganisationand binding affinity Thedevelopmentofconstrained peptides is an active andproductivestrandof research in thesearchfor ligands to inhibit D-helix mediated PPIs.Weperformedstructural andbiophysical studiesonBcl-2 familyproteinsusing constrainedvariants of theBH3 domainsofBID andBIM.Our structural investigations showedthatthe bound states of constrained andwild type peptides aresimilar (Figure1), with no enhancementininhibitory potency observed on theintroductionofpeptide constraints.SPR andVan’t Hoff analyses gave results consistentwithaninducedfit bindingmechanismand enthalpy-entropy compensation,contradictingthe hypothesisthatpre-organisingapeptideshouldenhance its target bindingaffinity.

Figure 1: crystalstructure of constrained peptide/proteininteractions(a) enlargement of Mcl-1/BID-MM (b) overlay of BID-MMand Mcl-1/BIM-WT, side chains fromBID-MMare showningreen,withBIM-WTsidechains in blue.

Developmentoftopological D-helix mimeticsscaffolds Identificationofeffective mimics of proteinsecondary structures that actasinhibitorsofPPIsremains amajor challenge.We designed andsynthesized foldamersbased on successive D-, E-and J-aminoacids, D/E/J- peptides, in which trans-2-aminocyclobutanecarboxylic acid(tACBC) promotes a hydrogen-bonded12,13-helicalconformation, hence mimickingthe D-helical conformation (Figure2a).Suitably functionalized D/E/J-peptides were shown to exhibitenhancedproteolytic stabilityincomparison to thewild-type D-peptideparentsequence fromwhich they arederived, and actaspotentand selectivepeptideinhibitorsofthe p53/hDM2interaction.

HIF-ĮSLQKLELWLRQZLWK³ELRQLF´SURWHLQV Theabilitytoincorporate non-natural functionality into bio-moleculesrepresentsanemergenttheme in synthetic biology. Creating such “chimeric” bio-molecules mayimpartnew functionalityand orthogonalpropertiesinto knownbio-macromolecules. Oureffortsinthisarea focussedonthe incorporationof ROLJREHQ]DPLGHVHTXHQFHV ZKLFK WRSRJUDSKLFDOO\PLPLFDQĮ-helix)intotruncatedHIF-Į sequencestogenerate “bionic”proteins(Figure 2b). Thesenovel peptide-oligobenzamide hybridswereshowtoefficientlybindp300withaffinitycomparabletothe parent peptide )LJXUHE )XUWKHUPRUHWKH VHOHFWLYLW\RYHUDVLPLODUĮ-helix mediated PPI(p53/hDM2) was increased compared to thatofthe non-hybrid oligobenzamidesequence.

75 main

Figure 2: design synthesisand testingof(a) D/E/J-peptidesasinhibitorsofthe p53/hDM2 interaction and (i) thenative D-p53 peptide and (ii) D/E/J-peptide helixmimetic (iii)Superimposition of thehelicalsegment of p53 highlighting thehelical backbone (inred), thehot-spot side chains(in green) andthe molecular modellingof thelowestenergy D/E/J-peptide conformer (inyellow) (b) “bionic” peptide-oligobenzamidehybrids.(i) peptide- oligobenzamidehybrids inspired by atruncated HIF-Į VHTXHQFH LL )OXRUHVFHQFH DQLVRWURS\ )$ competitiondatafor inhibition of p300/FITC-HIF-Į &-TAD forHIF-Į794-826 (orange) andhybrid 2 (black).

Publications Azzarito V.,RowellP., Barnard A.,Edwards T.A.,Macdonald A.,WarrinerS.L.&Wilson A.J. (2016) Probingprotein surfaces: QSAR analysis withhelix mimetics. ChemBioChem 17:768- 773.

LiuL.J., He B.Y.,Miles J.A.,Wang W.H., MaoZ.F., CheW.I.,LuJ.J.,ChenX.P., Wilson A.J., Ma D.L.,etal. (2016) Inhibitionofthe p53/hDM2 protein-protein interactionbycyclometallated iridium(iii) compounds. Oncotarget 7:13965-13975.

Miles, J. A. Yeo, D. J. Rowell, P. Rodriguez-Marin, S. Pask,C.M.Warriner, S. L. Edwards, T. A. &Wilson A. J. (2016) Hydrocarbonconstrained peptides–understanding preorganizationand bindingaffinity, Chem.Sci.,7,3694-3702

Burslem G.M., Kyle H.F., Breeze A.L., EdwardsT.A., Nelson A.,WarrinerS.L.&Wilson A.J. (2016) Towards"bionic'' proteins: replacement of continuous sequencesfromHIF-1D with proteomimetics to create functional p300 bindingHIF-1D mimics. Chem.Commun. 52:5421- 5424.

HewittS.H.&Wilson A.J. (2016) Metalcomplexes as "protein surface mimetics''. Chem. Commun. 52:9745-9756.

Grison C.M.,MilesJ.A., Robin S.,Wilson A.J. &AitkenD.J.(2016)An-helix-mimicking12,13- helix: Designed D/E/J-foldamers as selectiveinhibitors of protein-protein interactions. Angew. Chem.-Int. Edit. 55:11096-11100.

Funding We gratefully acknowledge TheUniversityofLeeds,EPSRC, ERC, andThe Leverhulme Trust forfinancial support of this research.

Collaborators External: D.-L. Ma (HongKongBaptist University), C.-H.Leung (University of Macau), S. Robinand D. Aitken (University of Paris-Saclay).

www.astbury.leeds.ac.uk 76 main Profilingprotein lipidation in parasites with chemicaltools

MeganWright

Introduction Thecellexertsprecise anddynamic controloverprotein function by modifying proteins,a process known as post-translationalmodification(PTM).One such PTMislipidation, where lipids are incorporatedinto proteins.Thisoftendirects theprotein to themembraneand is cruciallyimportant forproteinfunction. Because lipidationisanessentialprocess,there is interest in manipulatingthe enzymes andpathwaysthatcarry outthese modifications for therapy. However, globally identifying whichproteinsare lipidated in thewhole celland studying changes in acylation using biochemical methodsisvery challenging.

We have exploitedrecentadvancesinmassspectrometry-based proteomicsand bioorthogonalligationchemistrytodevelop chemical biology approachestoprofile lipidated proteins in differentorganisms.Inthe current project,weapplied our approach to analyse lipidationinthe Human AfricanTrypanosomiasisparasite, T. brucei.T.bruceihas acomplex lifecycle, involvingbothaninsect andmammalianhost.The focusofthe project wasonN- myristoylation: theattachment of thefatty acidmyristate to protein N-terminibythe enzyme N-myristoyltransferase (NMT).

Results We synthesisedalkyne-modified lipidprobes, includingmyristate analogue YnMyr,which were fedtoparasitesand metabolically incorporated into lipidated proteinsbythe cellularmachinery (Fig.1). Thealkynetag is small, inert andtherefore toleratedbythe biological system, butcan subsequently be detectedbyattachment of fluorophores or affinity labels viaclickchemistry. Experimentswere performedinbothbloodstream form (BSF,the host-infectivestage)and procyclic form (PCF,insect stage) parasites. We observed markedlydifferent profiles(Fig. 1), largelyreflectingdifferentialproteinexpression in theparasite, as well as incorporationof YnMyrinto stage-specificcomplex glycolipids. Usingpotentand selective inhibitors of the acyltransferase NMT, reductioninYnMyr incorporationintospecificproteinswas observed (Fig.1), indicatingthatthese are N-myristoylated andalsoproviding direct evidence that the inhibitors act on-targetincells.

Figure1:chemical proteomicstrategyand analysisofproteins by gel-based methods.Myristate analogue YnMyr is metabolically incorporated into proteins. Subsequentlabelling viaclick chemistry enables visualisationofproteinstaggedbythe probe, e.g. in different life stages(BSF,PCF). Inhibitorsof acyltransferase NMTinhibited YnMyr incorporationinadose-dependentmanner (right).

Tagged proteins were labelled with abiotinylated clickreagent,enrichedonstreptavidin beads, subject to tryptic digest andpeptidesanalysed viaLC-MS/MS. We usedlabel-free quantificationtocompare across samples: YnMyrvsMyr control; BSFvsPCF parasites; cells treated with andwithout NMTinhibitors.

77 main

Figure 2: chemical proteomic identification of acylated proteins in T. brucei.Plotofenrichment vs response to NMTinhibitor, enabling definitionofthe subset of acylated proteins thatare also NMTsubstrates. MG =contains N-terminal glycine (sequence motiffor NMTsubstrate); modified =siteoflipidation identified.

We subsequently compared thedatafromthese experiments with other recent published datasets fromour labs andothers, to yieldinformation on theprofile of lipidated proteins across differentprotozoan parasites. This analysis highlightedthe complementarity of different chemical proteomicsmethodsthathavebeendeveloped to investigate S-acylation, and showedthatacylationofsome proteinsisconserved across organisms,but that some acylated proteinsare unique to particular species.These datasets provideawealthofinformation on global lipidationpatternsinparasites, andsuggest that thesemodifications are both widespread andfunctionallyimportant.

In summary,chemical proteomicsmethodsprovideaglobal andunbiased waytostudy protein lipidationindiverse organisms. This contributestothe annotationofproteomes, andincreases ourunderstanding ofprotein modificationpathways andhow they maybetargeted fortherapy.

Publications Wright M.H.,Paape D.,Price H.P., SmithD.F.&Tate E.W. (2016)Globalprofilingand inhibitionofprotein lipidation in vector andhoststagesofthe sleeping sickness parasite Trypanosoma brucei. ACSInfect.Dis. 2:427-441.

Funding We thankthe University of Leedsfor start-up funding. Experimental work wascarriedout at Imperial CollegeLondon andthe University of York,and wasfunded by EPSRC, Wellcome Trust, andBBSRC.

Collaborators External: D. Paape, H. P. Priceand D. F. Smith (University of York); E. W. Tate (Imperial College London).

www.astbury.leeds.ac.uk 78 main Probing multivalentprotein-carbohydrate interactionsusing compact, polyvalent glycan-quantumdot andFörsterresonanceenergytransfer

Yuan Guo, Chadamas Sakonsinsiri,EmmaPool, WeiliWang,W.Bruce Turnbull andDejian Zhou

Introduction Multivalentprotein-carbohydrateinteractions are widespread in biologyand play acentralrole in viral/bacterialinfection,cell-cellcommunication andhostimmuneresponse.Theyinitiate thefirst contactbetween pathogensand target cellsthatultimatelyleads to infection. However, monovalent protein-glycan interactions areintrinsically weak,hence pathogens aredisplayed witharraysofglycanstheir surfaces, allowing them to bind efficientlytomultimericlectins on target cellsurfacesand exploitmultivalencytoenhance affinity.Understandingthe structure mechanismbehindsuchbindings is essentialtodevelop potent multivalent inhibitors. Amajor challengehereisalack of structural information forkey cell surface multimericlectins, dueto problems in solving such flexible,complex andmultimeric protein structure by crystallography. Althoughother techniques (e.g. isothermaltitrationcalorimetry,surface plasmon resonance) are powerful in providing bindingaffinities, kinetics,and thermodynamics, they cannot reveal keystructure information. Forexample, despite 20 yearsofextensive research worldwide,the complete structure of twovitally important tetramericreceptors, thedendriticcellreceptor DC- SIGN andits closely-relatedendothelial cellreceptorDC-SIGNR, remain unknown. Thesetwo receptors bind to virussurface mannose-containing glycansand facilitate theHIV/Ebola virus (EBOV) infection. Hencethere is an urgent need foranewmethodthatcan provide notonly quantitativebindingbut also structuralinformation to advancethisresearch field.

Results To addressthischallenge, we have developedanovelFörster resonanceenergytransfer (FRET) based ratiometric method to probe multivalentreceptorsDC-SIGN/R-glycan

(A) 2.0 2.0 (B) P (C) 0 M P 0.06 M 0PM 1.5 0.13PM 1.5 0.1PM 0.25PM P 0.38PM 0.2 M 0.5PM 0.4PM

a.u.) 1.0 1.0 0.63PM 0.6PM a.u.)

0.75PM I( 0PM

I( P 1 M 1PM 1.25PM = 0.5 1.2PM 0.5 1.5PM 1.4PM

0.0 0.0 = 500 550 600 650 700 750 500 550 600 650 700 750 O Onm nm

16 hQ (E) (D) QD QD 12 d Q 554 DC-SIGN +QD-Man h d /I 8 — DC-SIGNR +QD-Man x 626 I x CRD +QD-Man 4 CRD ' DC-SIGN +QD-ZW (i) (ii) (iii) 0 0.0 0.4 0.8 1.2 1.6 DC-SIGN DC-SIGNR C/PM Figure 1: (A) our approach to compact, polyvalentmannose cappedquantum dot(QD-Man) and probeits multivalent binding with tetrameric receptorsDC-SIGN/R via FRET (i)and tuninginter-mannosedistancevia thePEG linker length(ii) and diluting with inertDHLA-zwitterion ligand(iii). (B, C) directedexcitation backgroundcorrectedfluorescencespectra of QD-Man after binding to differentconcentration of Atto-594 labelled DC-SIGN (B) or DC-SIGNR(C). (D) plot of theapparent FRET ratioI626/I554 as afunctionofprotein concentration:the QD-Man-DC-SIGNbindingdatawerefittedbythe Hill’sequation. (E) ourproposedbinding models forDC-SIGNand DC-SIGNR bindingtoQD-Man.

79 main interactions. We have developedahighly-efficientcap-exchangemethodusing deprotonated DHLA-PEG-Man ligandand performing reaction in homogenoussolution.Thissubstaintially improved thecap-exchangeefficiencyand greatlyreducedthe amount of ligandrequiredfor complete cap-exchangeoverliterature methods by 20-200 fold.Compact (<10 nm), polyvalent mannose-QDconjugates (QD-Man,valency>150)were readily prepared. Importantly,the glycan valencycan bereadily tunedbythe linkerlengthand dilutingwitha spacer ligand (Figure1A).

TheQD-Manwas usedtoprobe multivlaent bindingwithDC-SIGN/R via aFRETbased ratiometicreadoutstrategy. DC-SIGNwas found to bind strongly to QD-Man (Kd ~0.3-0.6 PM) whichis>5000 fold tighter than monovalent CRD-mannose binding(Kd=3.5 mM). In contrast, DC-SIGNRbindingwithQD-Man wasrather weak:the resultingFRETsignalwas indistinguishablefromthatofmonovalentCRD-QD-Manbindingornon-specificinteraction betweenDC-SIGN andaDHLA-zwitterionligandcappedcontrol QD (Figure1D),indicating that DC-SIGNR cannotformstrongmultivalent bindingwithQD-Man.The FRET signal arising fromDC-SIGN bindingwas >60 fold stronger than that of DC-SIGNR.Thislevel of discriminationhas been unprecedented forthe twoclosely related, almost identicaltetrameric receptors.The differentDC-SIGN/R bindingpropertiesobservedbyFRETwas confirmed by QD-Man’s abilitytospecifically inhibitpseudo-Ebola virusinfection of only DC-SIGN-, butnot DC-SIGNR-, expressing cells.Astructuralmodel to accountfor such different QD-binding has been proposed:the CRDs in DC-SIGN face thesame direction, hence readilybindtoQD- Manmultivalently;whereas thoseinDC-SIGNRpointtodifferentdirections, making them unable to bind multivalentlytoQD-Man(Figure 1E). ThedifferentCRD orientationrevealed herein mayalsoaccountfor their differentvirus bindingand transfection properties.

Publications GuoY., Sakonsinsiri C.,Nehlmeier I.,FascioneM.A., ZhangH.Y., Wang W.L.,PohlmannS., TurnbullW.B.&Zhou D.J. (2016) Compact, polyvalent mannose quantumdotsassensitive, ratiometricfretprobesfor multivalentprotein-ligand interactions. Angew. Chem.-Int. Edit. 55:4738-4742.

Kong Y.F.,ChenJ., Fang H.W.,Heath G.,WoY., Wang W.L.,LiY.X., GuoY., EvansS.D., Chen S.Y.,etal. (2016)Highlyfluorescent ribonuclease-a-encapsulated lead sulfide quantum dots forultrasensitivefluorescence in vivo imaginginthe secondnear-infraredwindow. Chem. Mat. 28:3041-3050.

ZhangW., Wang F.,WangY., Wang J.,YuY., GuoS., Chen R. &Zhou, D. (2016) pH and near-infrared lightdual-stimuliresponsive drug delivery using DNA-conjugatedgoldnanorods foreffectivetreatment of multidrugresistantcancercells. J. ControlledRelease 232:9-19.

Wang J.N., Wang F.H.,LiF.Z., ZhangW.J., Shen Y.Y.,ZhouD.J.&GuoS.R.(2016)A multifunctional poly(curcumin)nanomedicinefor dual-modal targeted delivery,intracellular responsive release, dual-drugtreatment andimaging ofmultidrugresistantcancer cells. J. Mat. Chem.B4:2954-2962.

Funding This work wassupported by theWellcomeTrust (GrantNo: 097354/Z/11/Z)and theUniversity of Leeds.

Collaborators External: S. Pöhlmann (GermanPrimateCentre,Germany).

www.astbury.leeds.ac.uk 80 main

ASTBURY SEMINARS 2016

14th January Decipheringmolecularvirus -hostinteractionsbyanintegrated structural cellbiology approach Prof KayGrunewald, University of Oxford

4th February Solvingthe structures of protein-drug interactions using an optical analogue of 2D NMR: currentprogressand future prospects Prof DavidKlug, ImperialCollege London

3rd March Structural insights on small-molecule stabilizationof14-3-3protein-protein interactions Dr ChristianOttmann, EindhovenUniversity of Technology

7th April Developing betterinvitro models of theE.coliouter membrane -withthe help of neutrons Prof Jeremy Lakey, University of Newcastle

28th April Cheney FellowshipLecture Chemical biological modulationofRas signalling Prof HerbertWaldmann, MaxPlanck

5th May Protein foldinghomeostasis inthe Prof DavidRon,University of Cambridge

2nd June Thebacterialflagellar motor:remodellingananomachineasyou swim Prof Judy Armitage,University of Oxford

24th June AstburyAnnualLecture2016 Theuse of recent advances in electronmicroscopytostudy ribosome structure Prof VenkiRamakrishnan FRS, MRCLaboratory of Molecular Biology

10th November Targetingepigenetic reader domainsusing chemical biologyand medicinal chemistry Dr Stuart Conway,UniversityofOxford

1st December Heat ShockProteins-thebrainsofprotein quality control Prof Harm Kampinga,University of Groningen

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