The Structure of Pghl Hydrolase Bound to Its Substrate Poly&#X2010

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DOI: 10.1111/febs.14688

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Citation for published version (APA): Ramaswamy, S., Rasheed, M., Morelli, C. F., Calvio, C., Sutton, B. J., & Pastore, A. (2018). The structure of PghL hydrolase bound to its substrate poly--glutamate. FEBS Journal. https://doi.org/10.1111/febs.14688

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Download date: 03. Oct. 2021 This article is protected rights by All copyright. reserved. 10.1111/febs.14688doi: lead todifferences version between this and the of Pleasec Version Record. been and through typesetting, proofreading whichmay thecopyediting, process, pagination This article undergone has for and buthas accepted been not review publication full peer Accepted Date : 30 Revised Date : 05 Received Date : 14 1 The Randall Centre &Cell for Molecular Biophysics, King’s Keywords: Accepted ArticleSneha Ramaswamy 3 UK DementiaLondon,Institute ResearchLondon, at9RT,United College SE5 King’s The structuretoThe bound itssubstratehydrolase PghL of poly Running titleRunning Poly 4 Department ofChemistry, 2 The Institute, Wohl London,London King’s College(UK) - -

- - Aug Oct Oct - glutamate, capsule, PGA 1 $ *To whom correspondence be should addressed [email protected] , Masooma Rasheed, Masooma

- - - 2018 2018 2018 [email protected] : [email protected] The structure of PghL hydrolase bound to its substrate toits The structureofPghL hydrolasebound $ The authors have equally contributed 5

University of Sutton inhibitor, antiinhibitor, 1* , Annalisa Pastore Kingdom University of Milano,Milano (Italy University of 2 Pavia, Pavia (Italy)Pavia, Pavia ,3 - $ - microbial drug , Carlo F. Morelli

hydrolase, virulence, Staphylococci, biofilm , , +44 2078486423 , + 39 0382985545

, +44 2078485659

2,3 ,5 *

College London, LondonLondon, College (UK)

4 , Cinzia Calvio, Cinzia

- γ - glutamate ite this articleite this as

5 *, Brian

J .

This article isprotected rights by All copyright. reserved. specie Acceptedbacterial Some proteolysis. survive to polymer the allowing linkages, peptide α cleave only an recognition prevents linkage unusual The residue. next the of group amino α the with Glu a of γ position in group carboxyl chain side γ byjoined Poly Introduction of class this of mechanism catalytic the hydrolytic enzymes. on hints give and observed stereospecificity and substrate hexapeptide zinc its in PGA. effici dramatically and origin phagic of gene have stereospecificity We bacteria. resistant antibio to alternatives of development the for hope the dismantle can which characterised recently The health. human on influence profound a with tularensis F. anthracis, Article systems. defence host poly develop bacteria of spread mandatory. the become has of antibiotics to view resistance multiple in infections bacterial fight to strategies new of identification The Abstract PDB files: 6hrj, 6hri, 5onj NMR: nuclear magnetic resonance HPLC: dichroism Circular CD: Abbreviations: Article type : - 

s We used X used We -

ltmc cd (γ acid glutamic ih efrac lqi chromatography liquid performance high ae h ncsay isnhtc ahnr t prom h plmrsto reaction polymerisation the perform to machinery biosynthetic necessary the have

- - re zinc free, peptide

Original of the enzyme PghL (previously named YndL) fromYndL)named PghL(previouslyenzyme the of - ray crystallography to crystallography ray

linkages. This type of bond, classically found in glutathione, connects the the connectsglutathione, in foundclassically bond, of type This linkages. -

γ - mn te ahgn ta sil tesle i tee asls are capsules these in themselves shield that pathogens the Among - bound and complexed forms. forms. complexed and bound glutamic acid (γ acid glutamic ;

- and several and G) s ntrl oye cmoe by composed polymer natural a is PGA) EDTA:

 Articles - PGA hs eel dtis f h itrcin hc cud xli the explain could which interaction the of details reveals thus ;

- PDB: protein data bank acid Ethylenediaminetetraacetic capsules, are an attractive new direction that can offer real offer can that direction new attractive an are capsules, characteris

Staphylococcus Staphylococcus - PGA) capsules as a protectio a as capsules PGA)

solve d n eal h cevn mcaim and mechanism cleaving the detail in ed ent in degrading the long polymeric chains of chains polymeric long the degrading in ent d hydrolysis by classical proteases, which can can which proteases, classical by hydrolysis d

the three the

; IPTG: h poen rsalsd ih a with crystallised protein The tics, particularly in cases of multidrug multidrug of cases in particularly tics, tan. These strains. ; r - isopropyl dimensional pm:

It has been demonstrated that several that demonstrated been has It

revolutions per minute ; - n from external insults and/or insults external from n β γ - -

PGA D

huad o glutamates of thousands r motn pathogens important are structures of the enzyme the of structures B. subtilis subtilis B. - thiogalactopyranoside : poly  - G hydrolases, PGA - γ - encodedbya glutamicacid .

γ - PGA  B. - ; ;

This article isprotected rights by All copyright. reserved. surface. bacterial the to it linking and machinery biosynthetic the from γ growing the severing by acts normally that MEROPS), in (T03.023 genome stereospeci no with manner ( database peptidase γ the to belonging classes three known, currently proteases. common ( government Much arrest ( maturation ofphagosomal where macrophages, γ isolate of composition stereo γ unusual the by layer synthesises ( attachment covalent of without bacteria surrounds composed polymer heterochiral a secrete ( infections some γ uses also negative positive Gram the for both established extensively been has health human on impact significant a in resulting surveillance, immune host from bacteria protects γ that capsule a PGA of formation the allows surface cell outer the on chains polymer linking of capability archaeaand a fe Staphylococcus b particularly, microorganisms, soil recognition viral from receptors soil protects host shielding by infections phage thus prevents that barrier physical a creates also and surroundings metals, toxic from several both microorganisms including compounds, cationic chelate γ defence: to related mainly γ of chains long secrete and Accepted14 13, Article , 15 , γ - - F. PGA from this organism ( organism this from PGA PGA was first discovered as a component of the the of component a as discovered first was PGA ). This aim is thwarted by the polymer structure itself, which confers which itself, structure polymer the by thwarted is aim This ).

- tularensis, tularensis, PGA to evade host defences and its presence might be linked be might presence its and defences host evade to PGA 10 D 100% a w eukaryotes, produceandw eukaryotes, exploitthepolymer ( and 10 - defence 1 MEROPS in T03.001 2.3.2.2; (EC family (GGT) transferase glutamyl

). The majority of γ of majority The ).

γ 7 γ Bacillus - - ) both representing major biological both threats - glutamyltranspep PGA - . tularensis F. ) G dgaain eurs pcfc nye ( enzymes specific requires degradation PGA l plmr (γ polymer Glu , which hydrolyse γ hydrolyse which , ( 3 )

. The role of the γ the of role The . research is aimed at preventing or destroying or preventing at aimed is research ficity (1 ficity

appears to play a role in role a play to appears eicto ad osnn. erto o γ of Secretion poisoning. and desiccation - Hwvr a uh agr ubr f raim, including organisms, of number larger much a However, . - G hs hg water high a has PGA PGA. ( 1 8 a b dsigihd Te is cas nlds enzymes includes class first The distinguished. be can ) ). . Genes for γ for Genes . 1 6

2 oye. h lc o dt dpns n h iaiiy to inability the on depends data of lack The polymer. ). CapD is a GGT a is CapD ). n irba lf te ucin eetd y γ by exerted functions the life microbial In ) , possibly linked to its intracellular expression in host in expression intracellular its to linked possibly , t o ecuiey o te genera the of exclusively, not ut - tidase - D PGA - G) oaety nhrd o h peptidoglycan the to anchored covalently PGA) - PGA from its amino terminal end in an exo an in end terminal amino its from PGA -

PGA capsule as a fundamenta a as capsule PGA - CapD ( CapD producing bacteria, including including bacteria, producing

- 1, 1, PGA biosynthesis are abundantly present in present abundantly are biosynthesis PGA D - 10

n L and - F. tularensis F. ). In contrast, the pathogen the Incontrast, ). 11 bopin aaiy n te blt to ability the and capacity absorption - ) like enzyme present in present enzyme like

. No information is available on the the on available is information No - l ioes (γ isomers Glu Bacillus B. 2

( anthracis ). 5, 5,

phagosomal escape and/or escape phagosomal 6, 7,

anthracis - 8 1 G it te cell the into PGA the the , 9 ,

6 and for the Gram Gram the for and ). l virulence factor virulence l to ). -

DL γ epidermidis S. Among those those Among S. - persistence Streptomyces

PGA capsule PGA

asl. The capsule. resistance to resistance B. anthracis B.

B. anthracis B. - - epidermidis PGA chain chain PGA PGA) that that PGA) E - PGA are are PGA nzymatic nzymatic - type

of - , ,

This article isprotected rights by All copyright. reserved. cut efficiently to able and active enzymatically of subtilis treatment the B. for therapeutics as use recombinant the of structure the solved have we infections, potential recalcitrant their exploring at aimed ultimately infections caused by by responses immune of development appropriate develop to ( neutrophils organisms infected allowing models, γ of lack anthracis, their PghZ proteins These PghP. to homology at efficient were YoqZ and 41 and identity to found were YoqZ, and YndL YmaC, PGA. ( able only extraord zinc ( PghP bytypified is enzymes (2 L large superfamily o member γ (1 neutrophils by bacteria of killing and phagocytosis enhances γ the of degradation 23 - PGA degrading enzymes is represented byrepresented is enzymes degrading PGA Accepted Article– 5 kDa), kDa), 5 ). This specificity has been suggested to be due to the stereocomposition of tothe stereocomposition due been tobe suggested ). Thishas specificity - idn ezm ietfe i a in identified enzyme binding localisation , respectively ,

- With the aim of understanding the activity and stereoselec and activity the understanding of aim the With γ how Understanding glutamate rich (200 rich glutamate nrl efcie n fiinl dgaig h pol the degrading efficiently in effective inarily -

PGA synthesis has been shown to drastically mitigate bacterial virulence in animal in virulence bacterial mitigate drastically to shown been has synthesis PGA

o agt γ target to F. tularensis F. W hd rvosy eosrtd ht h rcmiat rti i fully is protein recombinant the that demonstrated previously had We .

te HP csen, histidine (cysteine, CHAP the f rbby cig ewe to D two between acting probably (18)( , 0 1, 4 15 14, 13, 10, 6, -

54% homology) homology) 54%

teaetc derivative therapeutic a in prophagic regions the of 4.0 i MEROPS) in C40.005 . Their genes are likely derived from integrat from derived likely are genes Their . - γ PGA ca PGA - - DL PGA

and - G, hl te are they while PGA, - Recently, four unannotated unannotated four Recently, – producing pathogenic bacteria.producing pathogenic psule of psule S. epidermidis, S. 450 kDa) fragments and D and fragments kDa) 450 p - PGA oly γ . n t In ). - ( DL 2 -

γ ). The authors also demonstrat also authors The ). were thus renamed by homology PghB, PghC, PghL and PghL PghC, PghB, homology by renamed thus were - - erdn ezms ok s n motn goal. important an is work enzymes degrading g - B. anthracis B. PGA lutamate lutamate e og em sc rsls il otiue o the to contribute will results such term, long he B. subtilis subtilis B. Te eobnn ezm hdoye γ hydrolyzes enzyme recombinant The . share with share f PghP of

erdto tu hglgtn ter functional their highlighting thus degradation B. subtilis enzymatic degradation of the γ the of degradation enzymatic B. subtilis subtilis B. - l rsde ( residues Glu h

 ineffective against the the against ineffective ydrolase of ydrolase - PGA

- with high concentrations of purified CapD purified of concentrations high with - ie nye a a ol gis persistent against tool a as enzymes like eedn amidohydrolases/peptidases dependent

at pae ( phage natto PghP

genome ( PgdS ( PgdS 2

- ). mr no ml oioes u are but oligomers small into ymer glutamate rich small oligopeptides small rich glutamate B

high

. We We p 9 2, 21 20, 19, subtilis subtilis hage; M86.001 in MEROPS), aMEROPS), in M86.001 hage; p

( oly now characteris now 3 sequence similarity sequence 2 ed prophages, as judged by judged as prophages, ed , 1 , ). tivity of of tivity ed that recombinant YndL recombinant that ed -

2 g 4 2 lutamate lutamate , 1 , . hs ezms are enzymes These ). gene products, YjqB, YjqB, products, gene 5 ). The third class of of class third The ). PghL hydrolase from hydrolase PghL . anthracis B. ). A second class of class second A ). γ - - PGA hydrolases PGA PGA capsule or capsule PGA d B. egradation), a a egradation), ed further the further ed

anthracis - G into PGA (27

capsule

In - 37%

B. γ ) -

This article isprotected rights by All copyright. reserved. in not distinguishable 24 residues) GluGlu) weight 2 fragments column determine We Char that suggesting respectively, °C 55 and °C 53 of phosphate in points melting with similar very EDTA the of stabilities the and spectra CD The EDTA. nati The ion. metal zinc a is homologue PghP suggested buffers phosphate respectively and Hepes in unfolding of ( temperature nm apparent 222 and 208 at minima with structure rich helical is protein the that confirmed (CD) dichroism exclusio size analytical from judged as temperature room at monomeric is protein The stability. and folding its for PghL characterised first We Characte Results provide insights the basis for into specificity its interaction the of details resolution high offer results Our of presenthe in andisolation in PghL both crystallised enzymeand this cleavagepropertiesof A

the

Accepted Article γ ).

, if change - h

PGA characteris

Longer acteris

reaction

only present,

species

derivatis

( remained

ris

Figure by the higher content of a of content higher the by and solved the structures of a zinc a of structures the solved and composed the

upon

ation PghL of stability ation ( oligomers products Figure

products

mixtures, zinc is not structu isnot zinc ed and from

prolonged ation.

2 of

the B

distinguishable were e rti (s oul epesd n prfe) a tu tetd with treated thus was purified) and expressed solubly (as protein ve

- 1 the the of E

C appeared

γ ) During released composed two

- . However, the re enzymatic

thus higher PGA Peaks progressively -

on ealppiaew epce htPh cud oti a contain could PghL that expected we metallopeptidase bound incubat

confirming

more hydrolysis

the gradually molecular attributable rally important by

in ion

activity  PghL, course

the glutamic -

rich structure after heating (data not shown). Since the Since shown). not (data heating after structure rich (data

two reduced

chromatograms

action is irreversible is indicatinaction that

from

weight the products

of not of to - to

stably folded. The spectrum is typical of a of typical is spectrum The folded. stably free

PghL

acid

PghL reaction .

longer the

the shown five to ,

background zinc

n chromatography ( chromatography n

short

indistinct residues reaction

and

glutamic

). - oligomers an

loaded and loaded

was -

Free

treated and untreated proteins were were proteins untreated and treated

wi oligomers endo the Figure thin

, f PghL of

were monitored

glutamic low envelop

enzyme - (

hydrolase Figure acid the

- (up initially molecular 1B

first

- (γ

PGA ih h sbtae and substrate the with residues

to . h poen a an has protein The ).

- of

g aggregation, as alsog aggregation, as acid GluGlu Figure f 3C n 5 °C 55 and 53°C of stereoselectivity. 2

b 6

eight E

y the

(22).

h ). enzyme complex. enzyme observed

HPLC

was

(

The Figure high weight

glutamic 1A were U and

not

sing pattern

). Circular Circular ). upon molecular

detected

( 2 -

Figure γ clearly Glu D higher -

PGA ). n

acid pre

did To - At  ce γ - - -

This article isprotected rights by All copyright. reserved. zinc apo between Zn The solvent. the to exposed core the of surface hydrophobic six by interleaved topology, seven a structure: structures. native and apo the in observed are ions and ion, zinc one contain also complex the and protein native The water. by hydrated others ( PghP the of structure crystal the using of resolutions at replacement molecular by determined of structures crystal The enzymeThe structure glutamic residues acid only. γ for PghL of stereospecificity the demonstrate material residual D of absence The detected be could ( configuration γ the and D contained dinitro glut free the fraction, individual cut a with membrane a using water against dialysis by recovered was fraction weight molecular verified and γ and GluGlu digestion. enzymatic by produced species weight molecular high the and fragments weight (data amount N

Accepted the as high as not is structure native the of shell outer the in completeness the that ote Article -

f o 3 of off - PghL the residues involved in Zn in involved residues the PghL

bound (native) structure. not The stereospecific The - ( 5 Table 1 Table of -

 shown). fluorophenyl)

3 to 3 - enzyme, Glu , -

0 D. fe aii hdoyi o te rgnl γ original the of hydrolysis acidic After Da. 500

by and L and -  Glu - iue 3 Figure γ ) 3 and 3 ) -

. ratio its as , 1 lGu lgmrc rcin ecuiey otie guai ai i L in acid glutamic contained exclusively fractions oligomeric GluGlu

ncer antc eoac (NMR) resonance magnetic nuclear H - stranded stranded The asymmetric units for all three structures contain one PghL molecule molecule PghL one contain structures three all for units asymmetric The -

γ ltmc cd n h oioei fatos n is cuuain n the in accumulation its and fractions oligomeric the in acid glutamic - only -

lGu ee niiuly eaae b in xhne chromatography exchange ion by separated individually were GluGlu glutamic acid residues in a ca. 54:46 ratio ( ratio 54:46 ca. a in residues acid glutamic

 -

L 6 to 6 ity of PghL was established upon isolation of both the low the both of isolation upon established was PghL of ity B

γ - . ny n h hge mlclr egt rcin D fraction, weight molecular higher the in Only ). aiaie and valinamide - GluGlu

aie zinc native,  

- 7 together with the core the with together 7

rose with respect to the L the to respect with rose  he is sheet 2+ -  amic acid released was derivatis was released acid amic

eie ad ie hr 3 short five and helices ion is located near the ends of strands of ends the near located is ion - sheet while t while sheet

dimer 2+ ragd codn t a to according arranged

- co

re ao ad a and (apo) free

homolog and - analysed ordination are in the same conformation as in the in as conformation same the in are ordination

γ he - Gl hydrophilic surfacehydrophilic

u PB ceso cd: al ( 3a9l) code: accession (PDB

- γ by HPLC. While the starting material material starting the While HPLC. by

- gL s goua poen ih a with protein globular a is PghL 

GluGlu 1. - - sheet form a cleft. a form sheet enantiome

03 10

Å, 1. Å, helices spectroscopy  -

G cmlxd PghL complexed PGA trimer - G sbtae n o each of and substrate PGA  7 ed with the chiral Nα chiral the with ed -

1, PGA chains containing L containing chains PGA iue 3 Figure Å and 1.7 Å, respectively, Å, 1.7 and Å r ( r . h hlcs rtc the protect helices The

 66D:34L) ( 66D:34L) accumulated regions of the sheet aresheet the of regions 3,  2 and 2  , while the higher higher the while , 2, In the structure of structure the In A )  , -

4,  ltmc acid glutamic the γ the 4. The loops The 4. Figure 3 Figure  Figur -

molecular after 7, - GluGlu  sulf

- 5,

4 e were (2,4 24  C ate ate  /

γ ) ).  h 6 - - - .

This article isprotected rights by All copyright. reserved. this for reference a as used often and characterised well protein a 3cpa), code: accession eutropha R. 2.9 r.m.s.d. from protein hypothetical a identified hits following 1.2 r.m.s.d. and clos the which amongst proteins bacterial mainly are hits higher The carboxypeptidases. mostly ( nearbychains. side the to or zinc the to close sufficiently is molecule water ordered No ion. zinc the from away par to the away but far too by, is close former lie His144 and His77 way. equivalent topologically a in binding zinc in ( hexapeptide penta complex, the in while solution, crystallisation the from citrate sodium of contribution further hexa PghL, native In mode. bidentate a in Glu46 of oxygens N chain side the from contributions has and native of structures the both In comparisonZinc carboxypeptidases classical and coordination with by stabilised be to appears which of interactions theadjacent with Phe133. one conformations different two in structures. the of any in observed C LEHHHHHH the of residues N additional an is there structures complex the in and apo the In structures. three all for observed are 207 to 7 residues for density Electron strand between loop the in proteins. similar the with agreement in structure, 0.15 of atoms backbone the between Acceptedhttp://ekhidna.biocenter.helsinki.fi/dali_server/ Article e homolog PghP from PghP homolog e - oriain cus ih h car the with occurs coordination oprsn f h codnts ih h etr PB sn te ai server Dali the using PDB entire the with coordinates the of Comparison root with fold, similar a have structures three The Minimal structural differences are observed for residues 142 residues for observed are differencesstructural Minimal - 3.1

( Figure 5 Figure qs Z cr 1. and 12.2 score Z 2q7s, Å

o 18 eius codn t Dllt) ( Dalilite) to according residues 188 on Å depending on the chain) and an an and chain) the on depending

A ). Thus, although different, the two small molecules both participate both molecules small two the different, although Thus, ).

phage s ticipate in coordination, and the side chain of the latter points points latter the of chain side the and coordination, in ticipate  and 6

ΦNIT1 is of course the closest structure (3a9l, Z score 32.3 score Z (3a9l, structure closest the course of is ΦNIT1 - The side chain of the solvent exposed Arg190 is observed observed is Arg190 exposed solvent the of chain side The emnl a isre fr uiiain upss r not are purposes purification for inserted tag terminal γ

- -  0.19 PGA

 ad oiind t h eg o the of edge the at positioned and 7 .... 3.1 r.m.s.d. oy oye ao o Gu i te bound the in Glu4 of atom oxygen bonyl 1 atoms of His41 of atoms 1 thermal unfolding thermal ) resulted in a large number of hits which include which hits of number large a in resulted ) - Å complexed PghL, co PghL, complexed . -

terminal alanine residue visible; the last seven seven last the visible; residue alanine terminal hs ofrs ht ic os o mdf the modify not does zinc that confirms This N Å - A. tumefaciens A. formylglutamateamido . oie abxppiae (PDB A carboxypeptidase Bovine ).

- and His102, and the two carboxyl two the and His102, and mean behaviour of the apo and native and apo the of behaviour - coordination results from the from results coordination iue 5B Figure - square deviations (r.m.s.d.) deviations square - ordination of the Zn the of ordination

(2odf, Z score 12.7 and 12.7 score Z (2odf, - 146, which are located located are which 146,

) . -

hydrolase from hydrolase fe ti, the this, After aayi cleft. catalytic 

stackin γ 2+ - PGA

ion g

This article isprotected rights by All copyright. reserved. solvent. Any tail, crystallisation hexapeptide, weight crystallisation defined concentrations the strategy crystallised the Electron structureThe a of homology equivalent PGA His77, His40, involvement observe conserved bidentate location. carboxypeptidase structures ( 3.2 r.m.s.d. and 8.9 score (Z score lower much a with but appears also family, enzyme Accepted Article Figure 5C

crystals

polymer

approximately - additional degrading

His102,

The Glu45, d Molecular

electron

density

PghL mode

is to in

crystallisation. ). grown of

using

residues

much

of we (average Glu270

other PghP

which

medium His78,

and residue these

residues

overlap must activity PghP for density

combination A,

Dimensions screens lower. in

γ spans

Glu165 homologues a

with

PGA complex

0. trappe conserved which

accept hexameric if His103,

molecular

( carboxypeptidase well, and

Figure

His41 not

for

( M

the

23 This  which (from

- d

sodium PGA ). that a

coordinat

in whole the

for

other PghL)

bound

and Glu165 method

material with

and

residues

their

 ( γ 6

hexapeptide. weight 2 Molecular instance, - would - A shares

PGA PGA ). Glu165

acetate, ).

His102, length

classical

have

The hexapeptide, non e

sequential

Since

exploits

peptide from (200 the in overhang

200

also A, zinc

been

PghL with

of catalysis to

PGA

zinc - - Dimensions)

a it although

400 the 400

precipitants. alanine nd the

This

atom

the 5.0, is reported

was

His40,

medium

position, the

ion protein

similar kDa) kDa

highly the

which

high

specific

binds 5% the

observed

sits two

are the ) protein testified

His103

w/v contained C nucleation which

PghP

precisely and - must must unlikely

to degree carboxyl environment which terminus strongly In have

then

 the

carboxypeptidases

PGA the in packs and

(equivalent have by enables have

and

the catalytic in

contain

structure deleterious

- the sufficient

that

precipitation

and

conserved oxygens Glu45 their

sequence of structure been against

been

highly fact

the

 its 20% the (

Figure

hydrolysed

three

γ site

same trapped , that to

of -

use is

PGA

high there

quanti exposed

PEG

His41, obtained

effect and PghP also

and,

( - mutations between

Figure

dimensional at

potential Glu46

position

molecular 5B

is structural 3350 such very

from ties

spatially

head

upon and

). Glu46, clearly

during a to

from

of in new

The low - and

B are the the the

to Å

of as of as  ) a a - - ) .

This article isprotected rights by All copyright. reserved. conventional substrate and instance, replaced ( reported main His78,Thr80, hosts agreement the carbonyls of the structure PGA conventional PGA homopolymer including interactions carboxypeptidase occurs than This functional proximity positioned 2

Accepted3 Article Asn73,

substrate carboxyl

Ser80

tight for pepti chain a These chain

An A phosphate

through by to ll

of any Arg127,

recognition

anchoring

de role to groups with, Ser74,

extensive in of

six

nitrogen

result bovi quite

observed

Ile45, ( are

polypeptide residues groups polypeptide is Ser81, the

Figure other such PghL

chain in

residues

roughly ne

and

typically

zinc different the A) Asn144 His77, in

interactions at

ion Ser70, carb a

polypeptide

(data

Glu165 atom could

explain of network with

6 almost

way with hydrolysis and

positions in

D are

(distance whose

oxypeptidase the

). equivalent of

PghL

chains.

Thr79,

chain

the

Gly72, not

a of that and

explain the conserved not arou

hexapeptide the

very

(equivalent features complete

Ser81.

key

shown) oxygen Arg145

amides

the invo nd conserved

peptide of

hydrogen i of

between ch 2.1

Ser80, -

Gly103, unusual 3,

the around side ain: to γ 6.2±0.3 lve

Mutagenesis -

Å) provide

PGA observed A i .

residues

that -

specificity

atoms of 2, abolishment

occupy

The

instead

(3cpa) chain in are to Asn151 (

(

Figure

the

Ala105, of spacing: bonds 3.8 - His77, by

classical in

enzyme 1, PghL in

Å,

the

substrate

form scissile

PghL, carboxyl

Å. classical

( the in

in i+1, an residues Figure

compared

smaller

and are 7

which of the Intriguingly, of 6

Thr79,

residues C, space

L Gly146,

hydrogen

the

this - and Thr80 carboxypeptidases,

formed

as configuration.

structure Thr168 residues E site

the

specificity 7 ).

NH expected group

the ), carboxypeptidases,

all ) family roughly

dipeptide

. Ser80 i+2.

These

(position

In indicating

activity

are of to Val147,

throughout tyrosine NH between

carboxypeptidase

and bonds

PghP

of of the the This

thus

and (Arg145

distances interactions

equivalent for

PghP, Glu4

Arg171

enzymes for position (Gly

distance of

i) Glu165 Ser148 The into likely

was with allows

zinc

the that γ the and

- -

in the

where PGA Tyr)

the

 suggested enzyme

carboxypeptidases.

- the γ wild this in

and PGA and position to

peptide for and alanine in to PGA

where

in main anchoring

are

observed an

be rather

side

Glu5

PghL)

His77, the atom the 

A type

Leu180

- extended in Tyr248 fragment

involve PGA

side

(3cpa), is

active anchoring

backbone chains

extended excellent

substrate i+1. has they in than in

enzyme plays

and

Thr79,

mimic chains

rather the in

been to

with

d The

site

the the are for for

of in γ in γ is a a - -

This article isprotected rights by All copyright. reserved. inte tetrahedral forma to molecule water coordinated acyl an form intermediate to enzyme carbon carbonyl scissile the attacks directly either A) carboxypeptidase gl distal the of chain side carboxylate in The hydrolases carboxypeptidases. PGA by hydrolysis catalytic of the of step first and the for suggested been general have models Two in particular. carboxypeptidases of both mechanism catalytic resistance. caus without reache were specifically the nevertheless maximise is knowledge structural Detailed pathogens. by sustained infections bacterial recalcitrant against agents therapeutic into engineered be to potential hold enzymes resistant and small robust, these us, for Importantly genethrough horizontal tr that demonstrated the of member view, of point a evolutionary an From reasons. several for interesting are PghL, of ( species bacterial several structure in identified recently family the carboxypeptidase here described have We Discussion cleavage occurs PGA LD degradation. ( this within PGA feature 2

Accepted Article,1 -

1 PGA enzyme

) hydrolysis, the the hydrolysis, glutamate Taken together, together, Taken .

B. The high resolution structure resolution high The A is

mongst

cleavage subtilis test

d, the proposed treatment c treatment proposed the d,

e γ for n drc bceil et ad c and death bacterial direct ing According

presence G chains PGA this

osblt o developing of possibility . 

(

- Figure γ the L

- pghP ( hypothesis, - PGA Figure PGA

ste (anhydride mechanism) or acts indirectly through indirectly acts or mechanism) (anhydride residues these results provide significant new insight into insight new significant provide results these

reospecificity of PghL and and PghL of reospecificity of -

to ie ee oiiae rm hgs n sra t svrl bacterial several to spread and phages from originate genes like 8 hydrolases

ansfer( as

Arg171 8 our

ih a with ) B compared .

). Together

we interacting prediction,

2 ).

des

which atclr stereochemical particular s could

of PghL th PghL of igned

ould expose bacteria to the innate host defence system defencesystem host innate the to bacteriaexpose ould to

with

enzymes

firmly be the through ne a

His77, involved

ould thus be less likely to induce bacterial bacterial induce to likely less be thus ould

Arg171Ser Arg171Ser cas f hrpui aet ta target that agents therapeutic of class w

at anchors

that provide an important guideline to the way way the to guideline important an provide

the Thr79 rmediate (water rmediate

repor in cleave

side

determining

the

mutation mutant and ted here provide here ted

carboxyl  chain composition the the

- D Ser80,t tmt (l20 n human in (Glu270 utamate -

PGA and essential essential

completely a -

promoted mechanism). promoted

the

his potentially previous studies have studies previous

group

tested

or activation of a zinc a of activation the the

2 stereospecificity

. If this objective objective this If . residue  ). These enzymes These ). γ -

- γ LD specificity insights into the into insights PGA

- prerequisite of PGA specific specific PGA it

abolished for PGA the

- conserved producing important

polymer third

chains of of

of γ to   - - - - -

This article isprotected rights by All copyright. reserved. length. whole its spanning cleft, site active the Accepted to oligomers Intriguingly these trimer. and dimer of conversion complete obtain to required was concentration enzyme 3 after present still were they that slowly so reacted reactor not did shorter Oligomers incubation. h 24 after mixtures reaction the never is substrates rate; fast relatively a at occurs that enzyme the by hydrolysis as to reasonable γ of hexamer a structure, trimers. pentamers and hexamers of level tothe down oligomers of short formation through the hydrolysisshowed progresses thatPGA crystallisation. account willneed Future Accordingly, totake observation. this into studies we other or viscosity the perhaps that suggests This polymer. the of polydispersity the despite medium since degradat catalytic by generated been have must that hexapeptide contained that screens Dimensions Molecular the using crystals obtained readily we whereas succeed, Article class ofthis ofinhibitors enzymes. inhibitor an in enzyme the to primary non a and a product cleavage is it that indicate here reported data cleavage the but substrate, a be to assumed is hexapeptide bound the that account into take also should We ligands. other by displaced the glutamate, distal the zinc the of of stabilization pKa the below values pH at that, known is It complex. the in buffe crystallization the from ion citrate a by played be could coordination zinc in water of role the water not is that mechanisma t close sufficiently is molecule water structure PghL the In other. the one over favour mechanism to evidence definitive no still is there studies, experimental numerous Despite

r in the native structure, and by a carbonyl group of the bound gamma group bound ofcarbonyl the aand by structure, native inthe r theless recognized by the enzyme as enzyme bythe recognized theless We tried to crystallise PghL using using PghL crystallise to tried We it is highly unlikely that sufficient quantities of a hexapeptide would be present in the in present be would hexapeptide a of quantities sufficient that unlikely highly is it biophysical hs rsls r fly optbe ih h srcua ifrain i te solved the in information: structural the with compatible fully are results These γ -

G (200 PGA clearly slow enough to allow crystallization of the complex. The hexamer was hexamer The complex. the of crystallization allow to enough slow clearly ue ht h hxmrc opud ersns h shortest the represents compound hexameric the that sume rpris f h longer the of properties - - 400 kDa) under various conditions. All our crystals contained a a contained crystals our All conditions. various under kDa) 400 coordinating water is weakened and this ligand could t could ligand this and weakened is water coordinating - G i bud o h atv st o te nye I i therefore is It enzyme. the of site active the to bound is PGA - ideal substrate. This could sug could This substrate. ideal - , the hexamer has the right dimension to fit completely across across completely fit to dimension right the has hexamer the , mediated. - ie anr dslcn te aayi wtr s s sa for usual is as water catalytic the displacing manner, like , and can proceed, with slower kinetics, to to kinetics, slower with proceed, can and ,

o the zinc atom to participate to atom zinc the o

On the other hand, other in other hand, other the On γ

- a substrate, since substrate, a PGA directly purified from purified directly PGA

, hs ugss ht we that suggests This γ

however, however, - PGA peptides are unfavourable for for unfavourable are peptides PGA

gest that the hexapeptide is bound is hexapeptide the that gest

its presence was not detectable in detectable not was presence its no crystallographically defined crystallographically no than six than

ion of ion the reactivity of hexameric hexameric of reactivity the

in the reaction the in 0 hours reaction. hours 0 terpretations are terpretations

glutamic acid residues acid glutamic longer peptide chains, chains, peptide longer B. subtilis B. rpe a hexamer a trapped - PGA hexapeptide form hus easily be easily hus

, suggesting ,

dimers and dimers rdc of product but did not did but

possible: possible: A higher higher A

This article isprotected rights by All copyright. reserved. isopropyl of mM 0.2 by initiated was Laboratories Melford expression (IPTG; thiogalactopyranoside Protein PghL. bound) 8.33 with supplemented densit cell optical an to up grow to allowed and overnight The incubator shaking a in rpm. 37ºC at incubated medium, 2xYT 200 of 1L inoculate to used at was culture agitated and 37ºC at overnight incubated Limited) 100 Scientific with supplemented media 2xYT of mL Biolabs) pETSL from induced was from PghL of Expression Protein production Materials Methods and as targetingenzymesforthese ofspecificity changethe for and target design drug abolishes serine shorter the in activity. Arg171enzymatic completely of mutation that demonstrated we Accordingly, L of specifically stereospecificity hold to positioned strategically and chains side enzyme the between interaction by determined is stereoselectivity that suggest substrate stereospecificity. achieves enzyme the conventional of spacing shorter much the with extended for specific is spacing Their chains. side protein the that explain Theyissues. peptide was trappedcrystal L clearlyan inthe that showed which enzyme this of stereoselectivity the of establishment the is results our of aspect important An of crystallisation could kinetically further compete against degradation. with species the is it because

AcceptedB. anthracis. Article The γ . -

A single colony from a freshly transformed transformed freshly a from colony single A PGA substrate is bound to the enzyme through a network of hydrogen bonds w bonds hydrogen of network a through enzyme the to bound is substrate PGA structures of PghL and its complex with complex its and PghL of structures L - γ

- PGA.

the only possible degradation products are L are products degradation onlypossible the

. subtilis B. why these enzymes areenzymes these why oe nhrn sd can, uh s ht f r11 are Arg171, of that as such chains, side anchoring Some

 M of zinc of M B. subtilis subtilis B.

noig the encoding einn mutatio designing

sufficiently high affinity for the enzyme, such that the process the that such enzyme, the for affinity high sufficiently

Altogether, t Altogether, gL for PghL

sulf according to the reading frame previously identified ( identified previously frame reading the to according ate just before induction to express the native (zinc native the express to induction before just ate h ntok f neatos ob interactions of network The G chains PGA pghL his knowledge holds promise for promise holds knowledge his γ - - PGA γ that homologues its and PghL in ns -  PGA oligomer.

gµ of µg/µL - gene - OD y pep γ

- specific carboxypeptidases. We observed We carboxypeptidases. specific - γ ahr than rather PGA thus clarify a numb a clarify thus PGA U) t 8C vrih. el were Cells overnight. 18ºC at UK) , - tid

E. coli E. D otiig L containing in BL21(DE3) cells cells BL21(DE3) in 600nm 600nm -

es. The structures also suggest also structures The es. PGA producing pathogenic bacteria pathogenic producing PGA γ abncli D oim (Apollo sodium D carbenicillin - PGA chains but is in is but chains PGA

of 0.8. Bacterial cultures were were cultures Bacterial 0.8. of plate was used to inoculate 10 10 inoculate to used was plate - glutamates. Accordingly,glutamates. γ - D - ltmt residues glutamate -

PGA explaining the the explaining PGA evd ih the with served

using PghL as a a as PghL using (New England England (New er of important of er compatible clearly would -

how ( - the the 2 ith ith D 2 ). ). - ) -

This article isprotected rights by All copyright. reserved. column(Phenomenex). 4.60, 5μm x 250 18 RP Gemini a using (Jasco) package software ChromNav running PC a to interfaced LC Jasco a on out carried used. HPLC and resin exchange ion 1×8 Dowex valinamide, 1 Chemical determination theenzymatic reagents for of instrumentation and activity state transition unfolding. two a assuming analysis regression nonlinear by analysed were data The 1°C/min. of were rate curves denaturation r the in heating Thermal while nm 222 at buffer. ellipticity the monitoring by appropriate obtained the of by corrected spectrum was the baseline subtracting the and accumulated were scans multiple measurement, each recorded(190 mM 5 in and 6.0, pH at phosphate sodium mM 20 in μM 5 of concentration a at prepared were stocks PghL Protein purified similarlywild tothe Arg enzyme mutant SDS by verified was protein the of purity the and nm monitore was profile elution The Biotech). (Pharma Purifier HiLoad a onto EDTA. mM 300 8.0 pH imidazole using by eluted were PghL native and apo (Generon), resin affinity NTA soluble. minutes 45 for rpm 15000 sonicated and discarded J Avanti Coulter, (Beckman centrifugation by harvested - Acceptedfluoro Article

γ - PGA characterisation - The supernatant was loaded onto the gravity column gravity the onto loaded was supernatant The 2,4 Basn oiir 250) Sonifier (Branson - dinitrobenzene dinitrobenzene

- 5D4L ws rm at Bocec (oy, Japan). (Tokyo, Bioscience Natto from was (54D:46L) 260 nm) on a Jasco J Jasco a on nm) 260 TM elt wr ssedd in suspended were pellets . Apo PghL protePghLApo . 16/60 Superdex75 prep grade column (GE Healthcare) using an AKTA AKTA an using Healthcare) (GE column grade prep Superdex75 16/60 loaded and mL 3 of volume a to concentrated were fractions protein The

ee (ih 5 mM 150 (with Hepes 171S

- 4000 instr 4000

. - a otie b site by obtained was type. n otat o rvos eot ( reports previous to contrast In pre Sne’ raet, Nα reagent), (Sanger’s -

in fractions were further di further were fractions in

equilibrated with the resuspension buffer. resuspension the with equilibrated - and 715 spectrophotometer using a 1 mm quartmm 1 spectrophotometerausing 715 ument connected to a UV/Vis detector (UV detector UV/Vis a to connected ument centrifuged

a) t H .. h fr V D pcr were spectra CD UV far The 6.0. pH at NaF) 0 mM 20 0 mM 20 - pcfc uaeei ad xrse and expressed and mutagenesis specific

(B - Tris PAGE gel PAGE

cmn ole, vni J Avanti Coulter, eckman Tris -

- 26XP) at 4 at 26XP) - grade solvents from Aldrich were were Aldrich from solvents grade C, 0 mM 300 HCl, - C, 0 mM 300 HCl, d by the UV the by d alys - (2,4 , packed with 5 mL Super Ni Super mL 5 with packed 2 ) the resulting protein was was protein resulting the ) (NuPage 12% BisTris). 12% (NuPage ed against buffer containing buffer against ed - dinitro o C. The supernatant was was supernatant The C.

PC nlss were analyses HPLC ange 25°C ange -

- 5 absorbance at 280 280 at absorbance al a p 8.0, pH at NaCl, -

fluorophenyl) The His The al 250 NaCl, z cuvette. Forcuvette. z

- - - 4070) and 4070) 99°C at a a at 99°C 6P at 26XP) 6 - tagged

mM The - L - - - This article isprotected rights by All copyright. reserved. collected, were Samples addition. enzyme by initiated was reaction the and °C 37 at γ of solution A lyophilization. cut low m A stained withmethylene blue. subtilis wild of µg 100 53°C was point melting 5 of concentration protein a using 6.0 pH at NaF mM 150 and Hepes PghL 5 of of concentration scan Thermal B) 30°C. at protein a using 6.0 pH at (blue) phosphate, sodium mM 20 in PghL curve: Black PghL CD. by monitored native and (red) apo of spectrum UV activity Enzymatic Enzymatic activity gradientusing analysed and mixed were acid trifluoroacetic aqueous 0.1% and solution resulting heatin and septum the needle a through inserting by evaporated was acetone the of Most min. 45 for dark the in °C 70 at batha water in heated and was sealed tube septum.The a pierceable and perforated cap screw acetone in reagent mM Sanger’s 10 of ofμL 500 and solution 8.5 pH buffer borate M 0.1 of μL 400 sample, reaction of μL 100 Pre 2.18 (td, 2.49 1H), Hz, 6.5 γ 2.23 (t γ were:shifts ( software inmr the with out carried were analyses Spectra 1 H NMR spectra were acquiredspectra were NMR H - - Accepted ArticleGlu GluGlu: - - - f membrane off - column derivatisation with 1 with column derivatisation ore detailed investigation of the activity was carried out by HPLC. HPLC. by out carried was activity the of investigation detailed ore molecular weight fraction of the commercial material by material commercial the of fraction weight molecular 2.09 (m, 4H), 1.99 - γ ,

- J in GluGlu:

= 8.1 Hz, 2H), 2.09 = 2H), 8.1Hz,

δ 0 mM 100

4.12 (dd, 4.12 .

- type PghL or Arg171Ser mutant were incubated with 4 µL µL 4 with incubated were mutant Arg171Ser or PghL type

 te high the , PghL is a folded and relatively stable protein. A) Com A) protein. stable relatively and folded a is PghL 4.35 (dd, 4.35

M. The calculated melting point was 55°C. Blue curve: PghL in 5 mM 5 in PghL curve: Blue 55°C. was point melting calculated The M. Tris J J

= 7.5, 2.3 Hz, 2H), 2.42 (t, 2.42 2H), Hz, 2.3 7.5, =

1.90 (m, 2H). = 8.8, 4.6 Hz, 1H), 3.26 (t, 3.26 1H), Hz, 4.6 8.8, = - was followed on agarose gels as previously described (1). Briefly, (1). described previously as gels agarose on followed was C p 85 o 1 a 3°, eaae o 2 TAE 2% on separated 37°C, at h 1 for 8.5 pH HCl

- J 2.01 (m, 1H), 1.96 -

PGA in HEPES buffer at pH 7.5 and 7.5 pH at buffer HEPES in PGA = 9.1, 5.2 Hz, 1H), 4.29 (dd, 4.29 1H), Hz, 5.2 9.1, = in D in - fr ute 1 mn Atr oln, qa vlms f the of volumes equal cooling, After min. 10 further for g oeua wih γ weight molecular - fluoro 2 O

at 400 MHz on a Bruker400 Ultrashield Bruker400aUltrashield on MHz 400 at - 2,4

ee ie i a ye tb eupe wt a with equipped tube Pyrex a in mixed were - dinitrobenzene(Sanger’s reagent) - 1.77 (m, 3H). J J

= 7.3 Hz, 2H), 2.36 (t, 2.36 2H), Hz, 7.3 = = 6.5 Hz, 1H), 2.34 (t, 2.34 1H), Hz, 6.5 = - PGA fraction was recovered by by recovered was fraction PGA

www.inmr.net J

ultrafiltration using a 10000 Da Da 10000 a using ultrafiltration

= 9.2, 5.0 Hz, 1H), 3.98 (t, 3.98 1H), Hz, 5.0 9.2, = 0.1 M NaCl was stirred was NaCl M 0.1 A  ). fter removal of the the of removal fter M. The calculated The M. parison of the far the of parison

The J J γ

= 7.4 Hz, 2H), Hz, 7.4 = = 7.9 Hz, 2H), 2H), Hz, 7.9 = - PGA from PGA spectrometer. - 1 agarose and and agarose H chemical H by HPLC HPLC by J B. B.

= - This article isprotected rights by All copyright. reserved. 1 in grew crystals PghL Accepted reservoirPghL mg/ nL solution. of and mL 500 mad suite). PACT suite, JCSG+ suite, II PEGs suite, (PEGs Qiagen II,JCSG Mol from screens and Labtech) (TTP robot pipetting nanolitre Mosquito a using Dimensions), Molecular plates, crystallisation (MRC plates well sitting by out carried was screening crystallisation Automated Structure determination by HPLC analyzed was acid glutamic resulting the and HCl M 6 with hydrolyzed was fraction weight fraction weight Glu freeze was water of portion first The w mixture reaction The min. 20 for °C 90 at heating by inactivated was enzyme the reaction the of end the At Determination enzyme of stereospecificity analyzedHPLC. by ArticleAft h. 1 for bath water a in °C 40 at heated and sealed was tube the mixing, After added. were Nα of μL 450 and p The septum. in transferred was mixture the and temperature room to cooled then was tube The h. 24 for bath sand a in °C seal was cap.with ascrewtube The dried. (γ fragments weight by analyzed and fractions in collected was eluateThe M). 5 to (2 acid acetic of concentrations increasing and water with eluted was pad 200 exchangeionDowex1×8 resin of pad a on loadedand adwas reactionmixture the of pH The bycheckedHPLC.was analyze and reagent Sanger’s with derivatised er evaporation of most of the acetone, the solution was cooled under running water and and water running under cooled was solution the acetone, the of most of evaporation er - e by mixing 500 nL of recombinant of nL 500 mixing by e γ -

Each pool of fractions was redissolved in water (10 mg / mL) / mg water(10 in redissolved was fractions of pool Each lGu 8 m) a rvae by revealed as mg), (80 GluGlu -

plus, Morpheus, Structure Screen 1 & 2, Stura Footprint, PACT, Proplex, PGA) andPGA) Proplex, PACT, Footprint, Stura 2, &ScreenStructure 1 Morpheus, plus, after pre H wa H

was recovered from the dialysis tube by freeze drying. The high The drying. freeze by tube dialysis the from recovered was - - (2,4 P another column derivatisation withNα column as then dialys then as

s adjusted to 8.5 with NaOH and NaOH with 8.5 to adjusted s - lGu n γ and GluGlu - dinitro

yrex tube equipped with a perforated screw cap and a pierceable a and cap screw perforated a with equipped tube yrex  -

5 ammoniu - fluorophenyl) ed against water using a membrane with 3500 Da cut Da 3500 with membrane a using water against ed ed after addition of 500 μL of 6 M HCl, and heated at and105 HCl, heated 6M addition of of500μL after ed - Glu - dried affording a mixture of HEPES, γ HEPES, of mixture a affording dried - PghL at a concentration ranging between 14 and 20 20 and 14 between ranging concentration a at PghL 1 sulf m

γ NR n HL aayi. h high The analysis. HPLC and NMR H - lGu wr cmie sprtl ad freeze and separately combined were GluGlu)

L. rcin cnann te low the containing Fractions TLC. d by HPLC by d -

L crystallised in4 t, 0.15 ate, - - valinamide 0.15 mg/mL solution in acetonein solution mg/mL 0.15 valinamide (2,4

300 μL of 0.1 M borate buffer at pH 8.5 pH at buffer borate M 0.1 of μL 300 ecular Dimensions (Clear Strategy I & I Strategy (Clear Dimensions ecular - dinitro

. -

400 mesh in the acetateThethe form.in mesh 400  After 24 h digestion, the reaction reaction the digestion, h 24 After

- justed to 9.5 with 0.1 M NaOH0.1M 9.5 with justed to 5 oim citrate sodium - Each crystallisation drop was was drop crystallisation Each - - 6 days. Apo PghLanddays. native Apo 6 rp aor ifso i 96 in diffusion vapour drop fluorophenyl) in a P a in yrex tube equipped tube yrex -

- GluGlu and γ and GluGlu L wt n pH no (with - valinamide. - - - molecular molecular molecular molecular - off. off. - -

This article isprotected rights by All copyright. reserved. INSTM The authors of interest. noconflict declare interest of Conflict statement ( 2012 (to Pavia of University Biotechnology, d ( assistance.forthe beamline their staff Diamond thank We Acknowledgements System, Graphics Molecular (PyMOL program v.1.8.0.5, Schrödinger,LLC, York, New NY, graphic PYMOL the with drawn were in given are PghL of structures refined the for statistics detailed The using ( software out carried was refinement ( Crystallographic PHENIX 3a9l). code: accession (PDB PghP co the with produced was model This ( PHASER in ( program AIMLESS unit. ( dimensions space each inthe onecrystal datasets collectedfrom were 1. to collected were complex PGA Kingdom). United Diffraction, oscillation) 0.5° frames, (314 apo for Datasets detector. 6M PghL X flash to prior cryoprotectant as freezing nitrogen. inliquid used were solutions reservoir respective The 3350. PEG adjustment) http://www.instm.it/consorzio/accordi/0000000384.aspx) RE13556), Eclez Porm (2018 Program Eccellenza i -

Accepted Articlefor UK) (Didcot, Source Light Diamond the at collected were data diffraction ray

(1000 frames, 0.1° oscillation) 0.1° frames, (1000 aa ee rcse uig h MSL sfwr ( software MOSFLM the using processed were Data 33 3 . PghL . 1 the Italian Ministry of Education, University and Research (MIUR): DipartimentiItalian University Education,and (MIUR): Research of the Ministry ). The program MOLPROBITY ( MOLPROBITY program The ). a ) 2

= 7 n RFA (32) REFMAC and

) with the coordinates of the model produced in Swiss in produced model the of coordinates the with ) 38.7, -  Light Source for access to beamline I03andMX9495) number:(proposal beamline accessforLightSourceto -

PGA crystals gre crystals PGA 2 6 b ). Initial phases were obtained by the molecular replacement method replacement molecular the by obtained were phases Initial ). =

47.3,

were collected in collected were PghL

– Complete datasets for native PghL, apo PghL, native for datasets Complete c 2022) = 03 . Model fitting was carried out with the COOT v.0.8.2 v.0.8.2 COOT the with out carried was fitting Model .

99.9 Å for apo for Å 99.9 (294 frames, 0.5° oscillation) oscillation) 0.5° frames, (294

using beamline I03 which was equipped with a Pilatus3 Pilatus3 a with equipped was which I03 beamline using

w in w This research wasby supported This research Å, 1. Å, A.P & A.P - - - Regione riae fr bacteriophage for ordinates

Dept 0. 7

1 USA). Å and 1.7 Å resolution, respectively. All three All respectively. resolution, Å 1.7 and Å 34 C.C.  s - of . ) was used to check for structure validation structure for check to used was ) house using an Excal an using house

sodium acetate sodium , respectively ,

PghL) and one molecule per asymmetric per molecule one and PghL) Molecular Medicine Medicine Molecular

. group P2

2 4 ).This work was funded by a by funded was work ).This

, pH 5.0 pH and PghL and 2 1 2 5 1 ) and and ) 2 the Dementia Research Research Dementia the  ibur PX Nova (Oxford Nova PX ibur 1 al 1 Table

I1 ic peptidase zinc NIT1 , 5% , - with cell unit similar PghL a and PghL model ( model - scaled using the the using scaled Lombardia Booy and Biology & γ w - / PGA complex complex PGA v . The figures The .  28 - PGA, 20% PGA, , 29 native

, grant 30 -  s. ). ). - 8. 7. 6. 5. 3. 2. 1.

This article isprotected rights by All copyright. reserved. 3101 AcceptedIdentification Su, tularaemia. subspecies Oyston, Michell Biochim. protects Monoclonal Jang, regulation Fouet, capsule Green, Articlehttps://doi.org/10.1371/journal.pone.01 Microbial Calvio, Mamberti, 1091 Candela, References the articlefrom everybodyelse withcontributions wrote AP project. the managed and results the analysed ch AP and CM BJS enzymatically, project, protein the promoted and plasmids the provided CC structure; the and protein the produced MR Author contributions

J., -

1098.

J.,

Yang, A.,

B.D., plasmid S.L., mice

P.C.

T., Cho, Biophys.

and S.,

(2010).

tularensis

(2015). antibody and

virulence

Dean,

from J., of Deletion Battisti,

Prati, M., Mock,

in Fouet, Zhao, Francisellatularensis

Bacillus Acta. enhanced J.

Lee, R.E., γ P.,

Med. Genomes. against -

PGA generates M. gene L., D.,

A. of

Cremaschi, 1830,

H.,

Eyles

(2006).

Differential

the Kawula, Microbiol. Koehler, anthracis

expression.

Hydrolases lethal

Cha, the carried out protein characteris protein out carried

2804 Bacillus

J.E.,

poly an

Poly

K., toxin

T. P., 12 . Hartley T.M., attenuated

- (1985).

59,

gamma 30810 -

influence Chun, H. Virulence gamma Seppi, (1996).

anthracis activity

of , 1275

Banas, PLoSONE. Thorne,

. M.G., Phage

- Infect.

J., D - - C.,

glutamate 84 Infect.

strain . due glutamic of Hong,

capB

Determinants. J. Morelli.

Waters,

the Origin

C.B., Immun. A., to

Immun.

that homologue capsule

two

K., &

in acid

Ivins,

C.F., Zhang, ation, SR crystallised crystallised SR ation, E.,

bacteria. protects Bacillus Park, 49,

64, Prior, capsule Bacillus and

291 Infect. Galizzi, B.E.,

10:

J., 4928 J.

in anthrax -

7 J.L.,

Mol. (2007). anthracis

mice

and

Francisellatularensis of

Demonstration – Immun.

subtilis 4932

A.,

Bacil

Titball,

Microbiol. Rhie,

against spore

aracteris Genome Fabbi

lus plasmids

and solved solved and G. and e0130810. challenge. R.W.,

75, anthracis

M., virulent (2013).

d the ed 60, - 3089

Other Wide

and and

a -

16. 15. 14. 13. 12. 11. 10. 9.

This article isprotected rights by All copyright. reserved. AcceptedSpringer. Hamano Kimura 1467 depolymerase Scorpio, 1014 Anthrax Scorpio, Chemother. Phagocytosis Friedlander, Scorpio, University, PhD ArticleFleming peptidoglycan. glutamyltranspeptidase Candela, epidermidis Key Kocianova, Acad. negative Weiss,

- Dissertation, role 1020

Sci.

D.,

K E.

A.,

A., with selection 95 A., Y, of

T.,

http://scholarscompass.vcu.edu/etd/16/

Brotcke, (2009). . Fujimoto

S., – 51,

Chabot, J. A. poly Tobery, S 117

editor. Chabot, overexpression

Clini. and Recombinant Mol. A.

and Vuong, 215 (2006).

Virginia 104, γ

- The scre -

222 Microbiol.

A., Killing

Fouet, Invest. D., Amino S.,

6037

D., -

en C.,

Potential

glutamic

Poly

family, Henry, Day, Ribot, 2010.

Commonwealth identifies

Yao, Day,

115, - 6042. - A.

acid Capsule γ

of reduces W., -

57, Glutamate

T., Enzymatic

W.,

Y., Role is 688

(2005) acid W., Encapsulated

O'Brien,

homopolymers 717 Margolis,

Voyich, required and -

genes

694. Bacillus for Depolymerase. Hoover, in - 726

Friedlander, immune CapB

Bacillus

Capsule

D., degradation required

J.,

J., for anthracis

Vietri, in T., Fischer,

Bacillus Chan,

Pathogenesis - evasion

the occurring Degrading anthracis and

for

A. N., Antimicrob.

K.,

covalent

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Enzyme

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MEROPS graphical

Poly

Crystallogr. ct that

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A.G.W.

T., T., is D., K.,

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Sci. hydrolyzes N.D., Z. Glutamate. involved processing

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Appl. - polyglutamic

- 234

enzyme glutamyl

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Role acid Hidesaki, .

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Finn, 46, -

2382

Novel

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D624 zinc Cryst

iMOSFLM: in -

Encapsulated bacterial

2291. Gene, 80.722

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This article isprotected rights by All copyright. reserved. PDBRCSB Code Outliers (%) Favoured (%) statistics Ramachandran in RMSD Average B Other molecules Water molecules Ligand Protein of atoms Number R R Refinement Statistics CC(1/2) B Wilson (outer shell) of reflections of No. number reflections (outer shell) Total (outer shell) Multiplicity (outer shell), (%) Completeness I/σI(outer R (outer shell), (Å) rangeResolution (Å) dimensions Cell asymmetric unit Number of / molecules Spacegroup 1 Table Data Statistics free cryst symm Bond(°) angle Bond length (Å) Solvent Protein Overall AcceptedLigand Article c b

(%) a

(%) (outer(%) shell),

–

factor (Å X

- shell) factor (Å

ray data collection and refinementray datacollectionand statistics

unique unique non

2 ) 2

- ) H

40627 (3713) 20566 (1942) 0. Apo PghL (1.76 22.7(3.15) 4.0 (21.7) 15.75 2.0 (1.9) b a P2 c 99 (96) 79 0.0 29.32 35.74 18.32 19.85 17.25 = = = 1629 1867 6hrj 1.67 21.2 17. 213

98 36 1

99.9 1 0 4 47.3 38.7 (0.35) 2

11 -

- 1 7

1.7) 1.7 2

14679 (2333) 76651 (1872) Native PghLNative 18.67 (1.34)

(1.07 0.99 c 2.2 (37.6) 34.2 1.9 (1.2) = b a P2 85 (21)

0.0 24.65 19.79 13.61 15.21 11.71 = =

1636 1916 6 1.51 18. 1 100.07 238 98 42 1 hri 7.3 (0.63) 1 0 4 46.8 38.7 - - 2

09 1.03) 1.03

1 9

PghL 15.74 40956 (3757) 20613 (1933) 24.58 (3.98) 0.99 (0.91 2.4 (13.9) - - 2.0 (1.9)

b a P2 c 99 (96) 1.7 (1.76 PGA complex 0.006 27.08 20.87 15.35 16.92 14.34 = = = 1618 1902 5onj 0.79 18.9 15.7 255

99 56 1 99.3 1 0 2 47.7 38.6 2

)

1.7)

This article isprotected rights by All copyright. reserved.  curve: pH of scan Thermal B) WT of Chromatographyprofile 1 Figure AcceptedM. The calculated melti Article

Comparison of the far the of Comparison 6.0 using a using 6.0 PghL –

in 5 in

gL s folded a is PghL protein concentration protein PghL

Hepes monitored ng was point 53° - UV spectrum of of spectrum UV and - PghL. The concentration of the protein used was 0.05 was used protein the of concentration PghL.The

by CD. Black curve: Black CD. by 150

n rltvl sal protein. stable relatively and of

mM 5

C 

. NaF at NaF M.

apo (grey) and native PghL (black PghL native and (grey) apo

The calculated melting point was point melting calculated The

6.0 using a using 6.0 PghL

in 20 in

prot mM s mM ein concentration of 5 of concentration ein

) ie Exclusion Size A) odium phosphate, odium ) at 30° at )

55° C. C.

mg/ml. mg/ml. C Grey . C . at )

This article isprotected rights by All copyright. reserved. Accepted derivatis reage Sanger’s excess the to due is 5 Peak resolved. is compounds di authentic of mixture di the to assigned peaks of hepta Signals putative visible. clearly are hexamers to up to attributable peaks the h: 6 by After D) caused material. peak broad a weight from molecular emerge higher to oligomers attributable peaks other defined; well are trimers and dimers peaks(γ dimers small h: 1 After B) present. is reagent Sanger the only 0: point Time A) for: chromatograms HPLC time. over products reaction of appearance the 2 Figure Article ation. Peak 6ac - – GluGlu) and trimers (γ trimers and GluGlu)

PghL -

and octapeptide are distinguishable as shoulder peaks. E) After 24 h: only only h: 24 After E) peaks. shoulder as distinguishable are octapeptide and - catalyzed hydrolysis of γ of hydrolysis catalyzed companies peak 5 as an unidentified impurity. peakcompanies an unidentified 5as - -

tri , 4, tri (4), - tetra , - - Glu 3, tetra (3), -

- γ a - nd pentapeptide are visible. visible. are pentapeptide nd GluGlu) appear. C) After 3 h: peaks attributable to attributable peaks h: 3 After C) appear. GluGlu) - 2 ad penta and (2) - PGA . Representative chromatograms showing showing chromatograms Representative .

progressively degraded polymeric polymeric degraded progressively - etds 1 ue a reference as used (1) peptides ih h rtnin ie of times retention the with t sd o pre for used nt F ) hoaorm f a of chromatogram - column

This article isprotected rights by All copyright. reserved. 3: Nα PghL from fraction weight hydro dinitro Starting A) valinamide. the of hydrolysis derivatis acidic after obtained were Chromatograms F

Accepted 3 igure Article - lysis; 1 = L = 1 lysis; (2,4 - 5 to o te eutn guai ai wt Nα with acid glutamic resulting the of ation - fluorophenyl) -

dinitro –

trohmcl ucm o PghL of outcome Stereochemical - - Glu, 2 = Nα = 2 Glu, 5 - fluorophenyl) - L γ - - aiaie B) valinamide. G fe cdchdoyi; L = 1 hydrolysis; acidic after PGA - catalyzed reaction after acidic hydrolysis; 1 = L = 1 hydrolysis; acidic after reaction catalyzed - (2,4

- - L dinitro - valinamide. - 5 γ - - lGu rm nyai rato atr acidic after reaction enzymatic from GluGlu fluorophenyl)

- catalyzed hydrolysis of of hydrolysis catalyzed

- L - - valinamide. C) High C) valinamide. (2,4 - l,2 D = 2 Glu, - dinitro ape ad pre and samples - 5 - - - l,3 Nα 3: Glu, fluorophenyl) Glu, 2 = D = 2 Glu, - molecular molecular - γ column column - PGA - - (2,4 Glu, Glu, - L - - . This article isprotected rights by All copyright. reserved. Accepted light and pink protein the panel: Bottom ( right) PghL and middle) teal, deep (in PghL native left), 4 Figure Article  -

PGA shown in blue). Residues involved in zinc coordination are shown as sticks. as shown are coordination zinc in involved Residues blue). in shown PGA –

in deep teal and sodium citrate (FLC) in light orange (middle)and (middle)and orange light in (FLC) citrate sodium and teal deep in PghL  - 2Fo PGA in blue (right). ThePGA in maps

crystal stru crystal - Fc maps for apoPghL with the protein the with apoPghL for maps Fc ctures. Top panel: panel: Top ctures.

were contouredat 2σ. Ribbon diagrams for apoPghL (in yellow, (in apoPghL for diagrams Ribbon - γ - PGA complex structure (in light pink, light (in structure complex PGA

in yellow (left), yellow in

native PghL with PghL native PghL - γ - PGA in

This article isprotected rights by All copyright. reserved. server Dali the by superposed as grey) in spheres. 3cpa, ( code: white) in 3a9l, code: Å. in are Distances blue). in shown PGA PghL and citrate) sodium (FLC: teal) deep in (middle, PghL native yellow), in (left, PghL Apo sticks. as shown are involved Residues 5 Figure Acceptedhttp://ekhidna.biocenter.helsinki.fi/dali_server/) Article

–

Zinc co Zinc - ordination site ordination C . ) Superposition of PghL (in pink) and carboxypeptidase A (PDB (PDB A carboxypeptidase and pink) (in PghL of Superposition )

and and B c - omparison of PghL with carboxypeptidases with PghL of omparison ) Superposition of PghL (in pink) and pink) (in PghLof Superposition ) γ - PGA complex structure (right, in light pink) ( pink) light in (right, structure complex PGA . Zinc atoms are shown as grey or red red or grey as shown are atoms Zinc .

PghP

(PDB . A)  -

This article isprotected rights by All copyright. reserved. Accepted The alphaare carbons of PGA sequentially. labelled ion. the zinc involve the that Interactions and enzyme the with interactions the indicating hexapeptide the of Formula red. in shown is ion zinc The superposed. structurally first were structures two The PghL. as view same the in represented substrate the mimics B). that residue tyrosine a with interacting to compared as 120° by rotated is ion zinc The sticks. as shown are coordination hexapeptide in involved Residues blue. in shown PghL B) pink). (light elect the showing Figure Article hw i rd C) red. in shown 6

–

h srcue f PghL a of structure The o dniy o te eaetd; gL s hw i rbo representation ribbon in shown is PghL hexapeptide; the for density ron - γ -

G cmlx tutr wt te nye n ih pn and pink light in enzyme the with structure complex PGA Close

protein side chains, and chains, side protein - p n ntok f neatos Te iw s approximately is view The interactions. of network and up - ) abxppiae (ca (hw i grey) in (shown (3cpa) A Carboxypeptidase D) γ - G complex. PGA

are thus sequence thus are

mt a cnord t 1σ at contoured map Omit specific, are boxed. boxed. are specific,  - PGA

This article isprotected rights by All copyright. reserved. Accepted and symbols plus respectively. residues The above. row andbinding zinc in involved the in given are entries UNIPROT the to according numbering the whereas row, last the in indicated is study this in used construct PghL the of numbering 7 Figure Article

– eune lgmn o the of alignment Sequence  - PGA sequence PGA

 - G cevn poen from proteins cleaving PGA - specific interactions are indicated with red starsred with indicated areinteractions specific

. subtilis. B.

T he

This article isprotected rights by All copyright. reserved. Accepted of above. theenzyme isindicated hydrolase mutant PghL. of Glu3 and Arg171 between Figure Article

 8 - PGA from from PGA

–

The key role of Arg171Ser in in Arg171Ser of role key The

for 1 h at 37°C at h 1 for . subtilis B. 

- a tetd ih 0 ng 100 with treated was PGA

and

. B) D B) . separat  - egradation by wild by egradation PGA ed

on on

anchoring. anchoring. a 2% agarose gel in TAE buffer. TAE in gel agarose 2%

of either either of A) Close A) - type wild

and Arg and - - up of the interactio the of up type

171S or

Arg er

Identity 171S mutant er n