Journal of Cell Science td sesnilt nesadmtrmcaohmclculn n nrytasuto,adcudla onwteaist treat to therapies new to lead could and transduction, energy and coupling swing. mechanochemical or motor disease. rotation understand human kinesins force- of to providin to a essential bind direction potentially resembles which is myosin, the that of study cardiac state to several by a inhibitors, opposite output in small-molecule mechanical oriented motors to increases the structure response trapping in force-amplifying fit, cycles the induced mechanochemical by their with in begins changes stroke undergo Motors each that reveals strokes 1 Kull Jon F. cytoskeletal myosin proteins and motor kinesin by generation Force Commentary hs nlsshsrsle nfrhrisgt nomotor 2011). into al., et insights found (Good be review further can recent inhibitors in a kinesin small- in on resulted myosin, information discuss of Further has activator function. also an analysis We and the kinesins We strokes. whose the motors states. of crystal working and inhibitors the nucleotide molecule their of on different motors compare mechanisms in based and force-producing the motors possible undergo, the propose of of they motor structures cycles changes discuss the We regarding conformational mechanochemical dyneins. known the kinesins is for the the more than on is mechanism which Commentary force-producing for this of myosins, Kon focus and 2011; The Ho 2012; 2012). al., their al., al., et et in et Kon (Carter also 2011; structure presumably al., transduction et overall and energy the myosins, in of by and deviate mechanism generation kinesins dyneins the force superfamilies, the from of contrast, myosin By mechanism and motors. common kinesin a the the implying the within of organization their form sides conserved core and opposite to elements are same structural on way These the domain. site same by motor filament-binding the formed unexpected or in is the nucleotide- organized an – proteins elements, myosins revealed both structural and of 1996) kinesins domain al., the motor between et homology kinesin (Kull a structural of structure domain the crystal by first produced motor The is incomplete. force still how is of past motors understanding the our over but studied years, intensively 25 been have motors Cytoskeletal Introduction words: Key and cycles mechanochemical their on based transduction, central the force of motor twisting of or mechanisms How Distortion contraction. structures. discuss muscle crystal and we transport in induced vesicle Here observed assembly, this? spindle changes drives conformational do that force motors producing ATP, do hydrolyze myosins and Kinesins Summary 10.1242/jcs.103911 doi: 9–19 126, ß Science Cell of Journal ( correspondence for *Author 2 rniinbtenmoi T-ieadncetd-restates. nucleotide-free and hydrolysis and ATP ATP-like myosin between transition eateto elBooy ueUiest eia etr uhm C270 USA 27710, NC Durham, Center, Medical University USA Duke 03755, Biology, NH Cell Hanover, of College, Department Dartmouth Chemistry, of Department 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. 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ATP ATP Myosin mechanochemicalcycle ADP ora fCl cec 2 (1) 126 Science Cell of Journal Mechanical cycle M Force Force M . ADP . ADP . P i M . ADP P . P i i P i n D.Scn,the Second, ADP. and M P . ADP i ADP M . ADP Kinesin mechanochemicalcycle Chemical cycle ADP M . ADP M c - M M . P . ADP i -op hc neat ihtencetd n soitdMg associated and nucleotide the cell with interacts the and which include These P-loop, role. crucial assays a play pocket nucleotide-binding kinetic the with together studies. analysis, biological high- mutant studies and functional to by crystallography that coupled X-ray microscopy, approaches cryoelecton by experimental resolution analysis through structural 1) include (Box identified been wthIadsic I hc c as act which II, switch and I switch the through primarily ion, itntptwy fcnomtoa hnet te ein nthe through in linked regions other are to regions change switch conformational two of the pathways distinct of each in Movements kinesins in pathways structural Force-producing 2 1, in Movies or material respectively). (supplementary 2003), lever 3, 1993) and and al., al., 1996) et et Cohen, (Rayment (Yun and arm (Houdusse stalk converter coiled-coil the or by 1999) myosins al., linker Their et neck the (Rice by cycle. kinesins the different of in catalytic amplified regions and other domain the motor to transmitted during are movements angstroms small relatively several flexible by these pocket absence As or 1). presence the (Box and of sensing 1996) GQ(T/ for I responsible are al., are switch sequence et regions motifs so-called switch (Sablin other the conserved motifs function, two II highly protein switch P-loop, motor the more for to essential a addition In myosins, has S)xSGK(T/S). and kinesins P-loop In is motifs. protein and the common GxxxxGK(T/S), most sequence the (Walker consensus of motif the one A has Walker 1982), a called al., also et P-loop, The 1). (Box 1996) nknsn n ysn,svrlcnevdsrcua oisin motifs structural conserved several myosins, and kinesins In c ATP popae hymv nadoto h nucleotide-binding the of out and in move they -phosphate, M M Force . ATP . ATP ATP P i M . ADP . P i 99 nrse l,20;Hle ta. 2011). al., et Hallen 2006; al., al., et et (Rice Endres motors 1999; force- kinesin the for be step to producing thought is binding ATP whereas rdcstemtrfregnrtn cycle. force-generating motor cycles the chemical produces and mechanical the of protein; coupling motor molecular ATP-fueled simple a of cycles. ( mechanochemical Motor 1. Fig. oc-rdcn tpocr with myosin occurs The step the motors. force-producing in two shift the the between show cycles to here simplified cycles, ( A B a ysnad( and Myosin ) ehncl(et n hmcl(ih)cycles (right) chemical and (left) Mechanical ) -, b -and c popae,adtoloops, two and -phosphates, c C popaesnos(Vale, sensors -phosphate iei mechanochemical kinesin ) P i release, 2+ Journal of Cell Science uloiefe tt rgt.Mcouue() B n ci iaet()shmtcdarm r retdwt h iu n otelf.Intermedi left. the to end minus the with 2004). oriented al., are et diagrams (Pettersen schematic Chimera (C) using filament interpolated actin were and structures (B) crystal (A), between Microtubule (right). state nucleotide-free h c tl gen it oad h lsedi h D tt lf)adrttstwr h iu n bakarw hntemtrrlae D n bin and ADP releases motor the when arrow) (black tha distinc resembles end conformation minus a region releases the assumes microtubule-binding and toward B) The ATP rotates 2). (chain hydrolyzes Movie and head it material (left) other as (supplementary state (right) the al. ADP 2011) et whereas the al., (Heuston – et ( in state Eg5. Hallen (left) ATP-bound end 2006; or state plus al., 2011) the ADP et Hwang, towards (Endres or and ATP tilts (Lakkaraju state post-stroke (green) pre-stroke the stalk the represent Ncd to – The thought structures is crystal which conformation, motor-ADP stalk-rotated as different conformation same the in ikdi h T-iesae(et u tagtn n oae ihtelvram ovre oag)adS1hlx(le upon (blue) helix SH1 and (orange) converter helix arm, to lever corresponds the (magenta) with helix rotates ‘relay’ and so-called straightens The but 3). (left) Movie state material ATP-like (supplementary the (right) in end kinked plus the towards arrow) (black oadtepu n bakarw n ok notemtri h T-iesae(ih)(upeetr aeilMve1.Central 1). s Movie then (left), material associ state (supplementary are ADP (right) the motor in state the end by ATP-like minus ATP the the of toward in extends binding motor which and the (blue), ADP linker onto of neck docks Release the and of complex. angle arrow) the helix the of (black yellow; in rest end change the large plus a with the and here toward (red) shown I not switch is of movement and with structures, crystal in disordered frequently ieo h oo oana h -opadsic .I ieis this kinesins, opposite In I. the L11, switch on and 1), P-loop loop, the (Box as a myosins) domain in motor the is helix of relay side motif the as II an to to (referred switch leads which kinesins, the in disordered frequently Following domain. motor microtubule-bi The 1). Movie material supplementary (see states two the ( between filament. interpolated represented intermediate schematically an a onto and ( docked 3HQD), elements motors PDB cycles. dimeric (right, mechanochemical the state their of ATP-like during head motors one show the that of structures changes Structural 2. Fig. C ysnI na T rniinsae(D DL lf)sostelvram(yn itdtwrsteatnmnsed h oo nege ag changes large undergoes motor the end; minus actin the towards tilted (cyan) arm lever the shows (left) 1DFL) (PDB state transition ATP an in II Myosin ) a 4-L12- a -emns le ( blue. C-terminus, 6 a ,mgna neatwt h irtbl.Lo 1,ajcn ohelix to adjacent L11, Loop microtubule. the with interact magenta) 5, C B A Lever arm MyosinII Kinesin-14Ncd Kinesin-5Eg5 Neck linker P B i ADP iei-4Ndi ie ihtohas nsakrttdNdcytlsrcue,oeo h ed PB31,canA is A) chain 3L1C, (PDB heads the of one structures, crystal Ncd stalk-rotated in heads; two with dimer a is Ncd Kinesin-14 ) n D,tastoigit h uloiefe tt PB1F)(ih) copne yalrertto ftelvrarm lever the of rotation large a by accompanied (right), 1DFK) (PDB state nucleotide-free the into transitioning ADP, and ADP α Relay helix 6 Converter ysnAPMyosin–no-nucleotide Myosin–ATP Eg5–ADP c–D Ncd–ATP Ncd–ADP α SH1 α 4-L12- 4-L12- β β a -sheet -sheet α hlx helix -helix, α 5 5 Stalk

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DP Neck linker P i a 4 ATP ATP eedn rettosadlnts hsptwylnsthe links pathway This with interactions allowing lengths. sites, nucleotide- filament-binding different and and of nucleotide- interface number a orientations microtubule-binding in observed the dependent been of has and component 2) major (Fig. a is helix oc eeainb ieisadmois11 myosins and kinesins by generation Force hne nmtrsrcuedrn hi ylsaeilsrtduigcrystal using illustrated are cycles their during structure motor in Changes a ,i lopr ftemjrmcouuebnigcmlx u is but complex, microtubule-binding major the of part also is 4, A Eg5–ATP iei- g nteAPsae(et D I6,the 1II6), PDB (left, state ADP the in Eg5 Kinesin-5 ) Neck linker + + + P i ees n rniinit the into transition and release b set a;N-terminus, tan; -sheet, a fteknsn;i is it kinesins; the of 4 00 (right). 2010) , fkinesin-5 of t t states ate nding wings tly ated ds Journal of Cell Science 12 Box 1. Motor elements involved in force production ora fCl cec 2 (1) 126 Science Cell of Journal

The nucleotide-binding P-loop (green) shows The switch II loop (cyan) acts as a secondary c- the side chains interacting with the bound phosphate sensor by forming a bond between nucleotide (AMP?PNP, orange). Two water the main chain amide of the conserved glycine Back Front molecules (red spheres) contribute to the and c-phosphate of the bound nucleotide in the octahedral coordination of the Mg2+ (magenta). ‘closed’ conformation; the glycine has moved The P-loop conformation does not change back too far to form this interaction in the ‘open’ substantially with or without bound nucleotide. conformation. Essential structural elements of kinesins and myosins shown in kinesin-5 Eg5. Left, nucleotide-binding P-loop (green), bound nucleotide (AMP?PNP, orange), switch I (red) and switch II (cyan). Right, microtubule-binding a4-L12-a5 (magenta), a6 C-terminus and neck linker (blue) and motor N-terminus (yellow).

Kinesin and myosin switch I and II regions share structural and functional similarities with G-proteins. Switch I (red), a flexible loop with the conserved sequence NxxSSR, assumes different con- formations during the force-generating cycle, switching between open (left) and closed con- formations (right), as shown in kinesin-5 Eg5, allowing the motors to function as c-phosphate sensors. Specific interactions of each switch element with the nucleotide determine whether it is in an Left, Eg5–ADP helices a4 and a5 (magenta) block neck linker docking. Right, open or closed conformation (Geeves and Holmes, 1999), although some differences exist between Eg5–AMP?PNP a4 and a5 have rotated, allowing the neck linker (blue; along myosins and kinesins in the switch I open conformation. In the closed conformation, the side chain with a6 C-terminus) to dock onto the motor. The loop extending from the N- hydroxyl of the first serine interacts with the c-phosphate, and the side chain of the second serine terminal b-strand of the motor (yellow), the cover strand, has not been fully forms a bond to the bound Mg2+, moving towards the nucleotide from its position in the open visualized in crystal structures, but is thought to interact with the kinesin-1 neck conformation (Kull and Endow, 2002). In kinesins, the movement of switch I from open to closed linker, forming a cover neck bundle. occurswithachangeinitsstructurefromashorta-helix (left) to an extended hairpin loop (right). In switch II, a loop with the conserved sequence DxxGxE, the closed conformation is defined by the formation of a hydrogen bond between the glycine and c-phosphate of the bound nucleotide (Kull and Endow, 2002). This interaction positions the loop 1–4 A˚ closer to the nucleotide than its more variable open positions. When both switch regions are closed, a catalytically active Pi tube is formed (Kikkawa and Hirokawa, 2006; Sindelar and Downing, 2010) (Fig. 3), in which two key water molecules in the active site are positioned to allow a nucleophilic attack on the c-phosphate by one of them, leading to nucleotide hydrolysis (Fisher et al., 1995; Parke et al., 2010). Journal of Cell Science ta. 00.I diint h ei-olo rniino wthI switch of transition helix-to-loop (Parke the solved to recently KSP), was addition closed, as focus In are known 2010). II into al., switch also and et come (Eg5, I 5 switch has both kinesin but which of in clear, structure the crystal less a to is when region transmitted I be switch to pocket catalytic vice-versa. and the interface, microtubule-binding in nucleotide the n r bevdi h T-on tt.TeCtriu of C-terminus The state. ATP-bound the in are helix observed that are motor helix and in this movements the same kinesins the of by a in primarily governed Interestingly, regions be to generation. the appears force to for transmitted responsible are cycle. region mechanochemical kinesin the of I between fine-tuning switch for interface, communication microtubule-binding allowing in of the and pathway movements site also nucleotide-binding distinct that the which second, possible L8, a loop is provide containing it region microtubules, a binds to adjacent is kinesins helix to adjacent the is which of analogue, comparison ATP 1), nonhydrolysable the AMP (Box to states (bound ATP-like closed and ADP and open the between rca o oeet(lnye l,21) hra nC- helix in of movement whereas motors, domain, be 2011), terminal motor to first al., the the shown precedes helix et of been neck the has (Clancy motors, which kinesin movement terminal 1), for (Box 1997) crucial an al., with et members family (Kozielski kinesin helix In domain, first motor domain. the N-terminal motor of the N-terminus the of as strand well as domain, motor conserved losaeanme fcmo ehnsi etrs ohhave Both features. mechanistic a common motors of these suggesting number that a shows same, share pockets also catalytic of the myosin Comparison and 1998). kinesin is al., the et (Kull order ancestor structural evolutionary topological these common between their insertions distinct elements, has motors of family a substantial share seven-stranded domains a a six motor of cytoskeleton myosin composed identity, different and core kinesin common with sequence the interactions tracks, an and of filament Despite size, 1996). lack in al., et difference complete (Kull unexpected structure was with core almost overlap it to motor shown hydrolysis, myosin was structure the ATP crystal that kinesin by in first similar the powered when are both proteins are motor they myosin and kinesin Although myosins and kinesins between mechanochemistry 2). in (Fig. helical Similarities direction the minus-end of the rotation in a stalk trigger coiled-coil kinesins, and to minus-end neck C-terminal, appear plus In movements microtubule 2). the initial Fig. in the and core 1 motor packing N-terminal, the (Box a against direction In in linker this diverge. neck result the At movements kinesins of these 2010). C-terminal kinesins, al., directed and C- et plus-end N- (Heuston loops the motors the point, kinesin of C-terminal rearrangements and helix identical helix to of nearly terminal end nucleotide- C-terminal in the the in to resulting transmitted changes are conformational region way, of binding end this the In of 1. rearrangement with strand associated also is which core, helix following ,dsusdaoe htafc neatoswt h microtubule the with interactions affect that above, discussed 4, ti loesnilta hne ntemicrotubule-binding the in changes that essential also is It the from leading changes conformational of pathway The a hlcs he nec ieo the of side each on three -helices, ? a N)srcue hw usata oeeto helix of movement substantial shows structures PNP) nknsn scoet helix to close is kinesins in 4 a opc onit ml okto h motor the on pocket small a into down pack to 6 a n -emnlto N-terminal and 6 b srn .Frbt -emnladC- and N-terminal both For 1. -strand a olwn h -op shelix As P-loop. the following 2 a sfloe ytenc linker neck the by followed is 6 a lostelo region loop the allows 4 b a srn nbt h N- the both in 1 -strand ,tels ei fthe of helix last the 6, b set lhuheach Although -sheet. b setfakdby flanked -sheet b -strand a 3in a a b b 3, 4, - - idn oatnidcsacnomtoa hnelaigt the to leading change conformational of myosin a release contrast, induces By actin microtubule. to the binding from domain motor bound ellrrls o xml,i ieis h opbtenswitch between loop the helix specific kinesins, and their in to II adapt example, to For cycle roles. the of cellular actin parts in diverged myosin, kinesins, subtly have for factor. for exchange whereas nucleotide and, – a ATPase 2006), as motor factor, exclusively motor the Hirokawa, functions exchange of activator and each nucleotide an (Kikkawa a as in the importantly, as is more filament perhaps both different is act the What microtubules of similar. both the In very hydrolysis and function same, cycle. are the point next is themselves changes the which changes conformational for of binding at order arm ATP the actin, lever and motors, the state, from repositioning rigor myosin the occurs, in releases resulting lost, then then is ADP ysn hl eahdfo ci.Hwvr hsi o u to due not is this and However, microtubules, their actin. to in from bound detached points while while different kinesin, myosin, is, at – That ATP cycles compared. hydrolyze mechanical closely myosin are and they kinesin when similarities exist more mechanochemistry, differences their than in motors myosin and of those to (Vale, II somewhat structure differ and motifs similar 1996). I sequence very their switch myosin, a and and share kinesin P-loop, G-proteins the and cycling of Although to switches, regions 1991). al., bind molecular forms and et inactive GDP-bound which as (Bourne and forms transition G-proteins, function active GTP-bound movements they with and between as shared GTP II is hydrolyze active similar in switch elements similarity This and site conformations. of closed show I and presence open switch between the of in nucleotides rearrangements determined structures different crystal kinesin and ope oafregnrtn ofrainlcag,and change, hydrolysis, conformational and motor force-generating binding the of ATP from release a subsequent by ADP to followed of kinesins, state-induced coupled For is loss the 1B,C). which in (Fig. by another nucleotide domain, results one caused binding to out is respect microtubule being the myosin with but and phase kinesin motor, of of cycles the in chemical and in mechanical changes changes conformational substantial ioaa 06 it ta. 08 idlradDowning, and Sindelar 2008; al., an et Helix forms Nitta 2010). and loop 2006; helix this binding Hirokawa, that of microtubule suggested upon extension been has stabilized in It becomes disordered structures. frequently is crystal and myosin kinesin in loop analogous the than osre ciest eiusmk iia neatoswt the with Mg interactions bound families similar and make nucleotide motor residues site helix two helix active relay conserved the and the as myosins known the helix between a in have structure both conserved and are above), and that (described motifs II sequence switch and I in switch as well as P-loops ysn h orsodn opbtensic Iadterelay the and and In II II switch manner. switch between microtubule-dependent loop closing a corresponding end, the in plus myosin, ATPase the binding towards the ATP tilt activating direction. a the the minus induces and the In open then toward 2010). be tilted would Downing, motor II and switch kinesin-1 (Sindelar states, nucleotide-free tilt kinesin- and can directed ADP plus-end domain the which motor upon 1 fulcrum fixed a as acts oc eeainb ieisadmois13 myosins and kinesins by generation Force rmti omnmcaohmsr,knsn n myosins and kinesins mechanochemistry, common this From vntog usata ifrne xs ewe h kinesin the between exist differences substantial though Even P a i a terlyhlxo ysn) opL1 slonger is L11, loop myosins), of helix relay (the 4 n oc-rdcn oaino h ee arm. lever the of rotation force-producing a and om h rmr irtbl-idn ie and site, microtubule-binding primary the forms 4 a P Hrs ta. 06 ikw and Kikkawa 2006; al., et (Hirose 4 i 2+ ytemtradrlaeo h ADP- the of release and motor the by a utemr,cmaio fmyosin of comparison Furthermore, . nteknsn Fg ) l fthe of All 2). (Fig. kinesins the in 4 Journal of Cell Science DLn,20) y,moi;SI,sic II. switch SwII, myosin; Myo, 2002). (DeLano, ( bottom). 1II6, xli h seta oeo wthIi h aayi yl.Tesrcue eeaindb h -op;budAPlk uloie(AMP nucleotide ATP-like bound P-loops; the by aligned were structures helix The (cyan); cycle. SwII catalytic (green); P-loop the open; in I switch of ADP role essential the explain h ciest hthsbe rpsdt rvd nei ot for route exit an provide to proposed been has that site active the u snwcniee niey(asne l,20;Klmne l,20) omto fa of Formation 2009). al., et Kaliman 2004; al., et (Lawson unlikely considered now is but wthI vnmr n irpigalitrcin ihthe with interactions all opening disrupting and thus more conformation, been Mg even twisted-sheet has II this a switch filament if as induces kinesin of in open, release occur binding Therefore, could is structures. binding relatively microtubule crystal I with upon of ADP bound switch number a remain when in can observed even ADP affinity, that clear high is it kinesin, ro oAPhdoyi htrsl nacoe,hdoyi-optn tt.Sic SI ntecoe ofrainecoe the encloses conformation closed the in (SwI) I Switch state. hydrolysis-competent closed, a ‘ in a result forming that nucleotide, hydrolysis ATP to prior oeet htdsutthe disrupt that movements i.3 omto fatb ysic o T yrlssand hydrolysis ATP for I switch by tube a of Formation 3. Fig. interactions Mg the and nucleotide A the disrupting 10 and I almost thereby switch I between switch site, moves binding in rigor also nucleotide-binding during cleft and occur actin-binding to actin, predicted the to is causes which close, twist to This myosin (supplementary 4). structures Movie nucleotide-bound material the with al., core compared et the (Reubold of II rearrangement substantial myosin b a and structures, 2003) these of In state al., 2003). nucleotide-free et the (Coureux in V seen myosin is structures crystal kinesin relay in the between necessary. link is is tight helix, II myosin a switch relay arm, and the when helix lever reposition is and occurs to them domain functions hydrolysis converter and between actin connection ATP from the because detached that – so shorter, stronger much is helix 14 ehns htrmi nla:mcouueidcdADP microtubule-induced actin-induced and of unclear: kinesins motor structures in the remain in EM release steps that and two for crystal mechanism mechanisms possible with suggest comparisons kinesin and myosin sethsocre,rsligi oepoone twist pronounced more a in resulting occurred, has -sheet n tutrlfaueo ysnta a o e encaptured been yet not has that myosin of feature structural One tutrlaayi ftencetd-recnomto of conformation nucleotide-free the of analysis Structural 2+ ? exfrMoV si h aepsto o aht ihih hr the where highlight to each for position same the in is V) Myo for BeFx ? D,a ntencetd-remoi structures. myosin nucleotide-free the in as ADP, ora fCl cec 2 (1) 126 Science Cell of Journal B ysnVwt on D wie oiinof position (white; ADP bound with V Myosin ) P i ue ntemtr KkaaadHrkw,20;Snea n onn,21)(o) olwn T yrlss wthIundergoes I switch hydrolysis, ATP Following (top). 2010) Downing, and Sindelar 2006; Hirokawa, and (Kikkawa motors the in tube’ P i ueadoe h ciest,poiigaptwyfrrlaeof release for pathway a providing site, active the open and tube Eg5 SwIclosed Eg5 SwIopen A a -emns(aet) ( (magenta). N-terminus 4 P-loop P i P-loop ees nmoi.In myosin. in release SwII 2+ . ˚ SwI wyfo the from away SwI SwII α α c P A P 4 4 popae ro)(D WJ o;PB18,bto) mgswr edrdi PyMol in rendered were Images bottom). 1W8J, PDB top; 1W7J, (PDB arrow) -phosphate, i i iei- g ihbudAMP bound with Eg5 Kinesin-5 ) release. ees Yute l,19;SenyadHuus,21;Lia ta. 2012), al., et Llinas 2010; Houdusse, and Sweeney 1995; al., et (Yount release h wthIadI ein fteknsn n ysn neg tutrlchanges structural undergo myosins and kinesins the of regions II and I switch The c popaewudb nteoe tutr.Tp w rd lsd otm SwI bottom, closed; (red) SwI Top, structure. open the in be would -phosphate oe the cover uloiefe n D ttssosdniyi the in density the shows in A states (10–12 complexes ADP kinesin–microtubule and high-resolution nucleotide-free of images of microscopy Comparison that xtrue tsol entdta D ol eanbound remain would and observed structures, as ADP crystal II, switch kinesin that and many P-loop noted in the with be interactions should of because It route. exit Lwo ta. 04 aia ta. 09,csigduto this on doubt for casting route 2009), alternate al., An et theory. 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SwI D,sic n wthI completely II switch and I switch ADP, i ol o erlae hog hsroute this through released be not could SwI c popae ro)(D HD o;PDB top; 3HQD, (PDB arrow) -phosphate, P i ees htwudb consistent be would that release P i u ol looe pan up open also would but , P i c ees i h wthI switch the via release popaeo h bound the of -phosphate P ˚ ? i cryo-electron ) N o g and Eg5 for PNP utei the exit must b -sheet Journal of Cell Science rbesascae ihbignnrcsieo rcsie It processive. solve or to nonprocessive approaches being similar kinesin with used that associated appears have problems it subfamilies as myosin evolution, domains divergent and motor by of related realm are the that beyond extend myosins and kinesins of release to coupled directly be ADP. necessarily not would opening ml oeue htbn oseii iei rtiscould proteins as kinesin serve specific 1999). might al., to et bind mitosis, (Mayer that cancer in kinesins, molecules against the roles drugs that Small for essential was targets field their effective motors of the in because realization early An inhibitors Kinesin the in body Similarly, rigid helix myosin. a a of movement of to to Ncd, motor leads stroke linked the kinesin-14 in resulting which is power arm, lever helix, II and force-generating domain converter relay switch 2, the of (Fig. of the movement movement of arm above, arm. movement turn, lever lever in helical described the is, the rigid with which As contacts II, domain, hydrophobic myosin converter tight a contraction, the of in of involved case out of muscle the extends In arm powering rotation lever 3). II Movie myosin myosin by material conventional supplementary the force that as years produce many such for motors, known been has ehnceia yl nihsit motor into function insights – cycle the mechanochemical of activators non- and versus Inhibitors are processive filaments. that achieve respective strategies their to along of ways movement set processive proteins various similar in motor to a appear employed myosin evolved then but convergently and ancestor, common have a kinesin 30– from divergently averaging that evolved A steps 65 remarkable variable to doubly up highly and taking nm 36 and mechanism sites ‘neck-linker’ kinesin binding in the processively arm. of walk that to (Me lever to motor similar the and manner allow that regions VI a domain insertion myosin and to arm converter unique lever are the of the in unfolding compliance of the between Regions involving al., insertion mechanism et similar an a myosin the Clancy processive to dimeric (see coordinates point a the heads VI, motor, myosin linkers, between two on studies strain the neck Recent 2011). that of bind the suggested cycles to though been mechanochemical has motor transmitted acting al., It et dimeric heads, Khalil force, 1). 2008; al., the produce (Box et (Hwang 2008) and strand’ of ‘cover microtubule the heads in with together the extends both to linker allow neck simultaneously The to 2, (Fig. 1). 1999) length the Movie al., to et material domain Rice motor 1997; supplementary al., the et connects (Kozielski that undocking hand-over-hand stalk linker’ and coiled-coil ‘neck This docking flexible sequential contrast, a mechanism. the of by different By achieved is steps a movement al., multiple et II. processively Schaap taking utilizing 1993; a 1990), al., 2011) et move myosin al., (Ray with et protofilament microtubule Block motors a of arm along 1989; al., lever kinesin-1 that et (Howard extended dimeric to an to conventional thought nonprocessive similar utilizing is are motor, actin mechanism Ncd processive along a and V, move II myosin myosin however, Movie material to motors; both similar supplementary very Interestingly, 2, stalk, (Fig. and 2). stroke neck power helical myosin Ncd the the of rotation large a ial,i sitrsigt oeta h iiaiisbetween similarities the that note to interesting is it Finally, ´ ne ´ rye l,21)wt h w ed tpigaogactin- along stepping heads two the with 2012) al., et trey ˚ pr Rc ta. 01.I stherefore is It 2001). al., et (Rock apart a per ola to lead to appears 4 otemtn hntp fteknsn5Bm rti Eo and (Enos protein BimC kinesin-5 the of similar spindle remarkably phenotype are mutant monoastral effects the on cellular to a These ring-like to effects 1999). a al., attached et with striking (Mayer chromosomes mitosis in mitotic its arrested of were of array cells the because – cells interesting dividing compounds, five a especially the of taxol, one was but cells, Monastrol, from all mitosis. mitosis in in differed microtubules those on affects thus including that effects mitosis drug effects anti-cancer have Their used affected widely to microtubules. found that on were A not compounds that 1999). al., five cell-permeable chemical et identified (Mayer a for mitosis in of discovered screen inhibitors for compound screen a genetics monastrol, was kinesin in mechanism force-producing motor cells. the live potentially unraveling also in are motors useful kinesin processes. be given they microtubule-based microtubule for other – to known specific of Compounds by cells disruption expected the caused all to effects due affecting are side of drugs the than they reduce many rather thus that cells, potential could drugs, dividing in to offer antimitotic microtubules, specific tumor used compounds target during currently These which division over cell metastasis. advantages block or and formation function motor disrupt niioi acrteaetc tsitdtetre fnew the of With target motors. the kinesin specific shifted to it microtubules – from screens therapeutics cancer in antimitotic microtubules spindle of motility, sliding cells. its inhibits dividing and monastrol of crosslinking hydrolysis, Because ATP reducing state. two slowing force-producing in The that show conformation effects ATP-like, kinesins. a both in an the trapped 1X88) to ADP, resembles and and of thought analogues monastrol 1Q0B to state are bound (PDB ATP and Eg5 ATP structures 2010), to the crystal al., available of bound et Parke characteristic motors 2001; be are kinesin al., linker neck et the in (Kikkawa and II observed switch in helices changes also latter II These switch 4). of (Fig. tilting the including I, involve site, switch binding a in not also the transition monastrol to does by loop-to-helix distal it Binding motor a motor. the fit, in the in induced in it changes noted fold induces Although as new state. to a as ATP-like of referred formation the state, be in still ATP-like loop restructures can the monastrol the stabilizing by L5, in binding This loop Thus, loop 4). of 4). 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L5 the active al., the by the below near formed et just site is that formed pocket (Yan 1Q0B) newly ‘induced-fit’ a cleft (PDB to monastrol human bound nucleotide-binding of compound with structure the crystal complexed revealed a of Eg5 report kinesin- not the on kinesin-5 monastrol with does al., of clearer effects et but became The 5 (Maliga 2003). activity al., binding et ATPase microtubule DeBonis 2002; or its nucleotide inhibits with compete further and domain been motor has 2003). al., motors et compound (DeBonis kinesin-5 others of by the vertebrate specificity demonstrated The for 1999). that al., Eg5 et monastrol (Mayer kinesin-5 showed motility motility the kinesin-1 kinesin-5 inhibit on KSP blocks monastrol specifically of homologue monastrol of Tests effects the vertebrate motors. test to kinesin authors the on led and 1990) Morris, oc eeainb ieisadmois15 myosins and kinesins by generation Force and 4 h is ml-oeueihbtrt erpre httree a targeted that reported be to inhibitor small-molecule first The h icvr fmnsrlwsagm-hne for game-changer a was monastrol of discovery The kinesin-5 the to binds monastrol that showed studies Kinetic a ,addcigo h eklne gis h motor the against linker neck the of docking and 5, nvitro in u osnot does but , Journal of Cell Science eebe oc-rdcn T-on tt.Sproiino h rti hiswspromduigMthae fCiea(etre ta. 2004 al., et Eg5–AMP (Pettersen then Chimera Eg5–ADP, of aligning conform Matchmaker a first assume using and essent to be chain performed Chimera. activity, to reference was ATPase in thought a the in chains is analyzed et inhibited as which protein and and Cochran Eg5–ADP–monastrol 1), the ADP 2002; (Box designating to of conformation al., by bound closed Superposition et parameters still I (Maliga state. default motor, switch cycle the ATP-bound a hydrolysis cause of force-producing ATP thus formation a monastrol the the resembles by inhibit blocking induced also or changes could slowing Eg5–AT structural I and than The switch release, Eg5-ADP hydrolysis. on resemble ADP monastrol closely of inhibiting more effects in to structural monastrol it The of causes which effects transition, the loop-to-helix with short central consistent a the undergone has of motor true the ( also of 2010). 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Fig. 16 ora fCl cec 2 (1) 126 Science Cell of Journal a and 4 ? b N ytretrs hc ih xli h ekmcouuebnigb h oo on omnsrl oprdwt g nteATP- the in Eg5 with compared monastrol, to bound motor the by binding microtubule weak the explain might which turns, three by PNP set niaigta h wsigo the of twisting the that indicating -sheet, a ADP or AMP om noeigta lostenc ikr(L odc;temtr ihadce eklne r nepee orpeetthe represent to interpreted are linker neck docked a with motors the dock; to (NL) linker neck the allows that opening an forms 5 A L5 a Eg5–ADP n hnL ()ad() re ro] ln hsptwy g–D–oato olw Eg5–AMP follows Eg5–ADP–monastrol pathway, this Along arrow]. green (B), and [(A) L8 then and 3 L5 Eg5–Monastrol . PNP ( A Eg5–AMP rsa tutrso iei- g–D–oato PB10,18)so oato on oanwst near site new a to bound monastrol show 1X88) 1Q0B, (PDB Eg5–ADP–monastrol kinesin-5 of structures Crystal ) α 3 α 3 Monastrol . 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AMP closer conformation ATP somewhat closed for a slightly essential the shows is be into ispinesib might to transition which or thought I helix-to-loop conformation, switch monastrol ADP-like its the an either prevent time, in to same remains the bound and of extended At Eg5 dock. site of to interface, the helix linker microtubule-binding neck at the the and to kinesin-5 allowing propagated in monastrol are changes that binding both conformation, structural ATP-like induce Thus, the ispinesib Eg5–ADP–monastrol. in are resembling Eg5–ADP–ispinesib helices of II and switch linker L5 et that tilted (Zhang show the monastrol Superpositions 2012). as L8, al., L5 et loop Talapatra near 2008; cleft al., Eg5–ADP ADP induced-fit to same bound inhibits ispinesib the show at (Lad structures ispinesib microtubules Crystal to 2008). 2005), binding al., motor et al., slows and kinesin-5 et by release (Cochran monastrol Like ispinesib by molecules inhibition small Kinesin-5 by inhibition Kinesin 2. 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P N,sgetn httepeitdtitn fthe of twisting predicted the that suggesting PNP, i tt,cue ytesrcua fet ftecmon on compound the of effects structural the by caused state, P i rdcino ees,cnitn iha with consistent release, or production a and 4 a and 2 a ,adtedce neck docked the and 5, a ,ajcn oL5, to adjacent 3, b setis -sheet b -sheet T yrlssb ada ysni h rsneo ci,but actin, of presence the in myosin slows cardiac by hydrolysis ATP ttsi h iei yl h oncetd,o osbythe possibly or no-nucleotide, the – cycle also missing kinesin could the ADP the of it one in compound; about the states information by valuable the provide reveal inhibition potentially could motor 2010) of al., et mechanism (Wood GSK923295 with complexed ainnis atclrytoerssatt urnl used currently to their taxol. resistant as of such drugs, those important or knowledge microtubule tumors an with particularly patients have our for play outcome malignancies, the also to improving in might role and contributed clinical function of thus mechanism have motors sa Mra ta. 00.Tsso h piie compound, accelerates optimized it the that of showed Tests mecarbil, 2010). identify omecamtiv al., myofibril et or to that sarcomere (Morgan reconstituted compounds assay a for designed in screen myosin myosin cardiac high-throughput II activate the was a specific myosin in cardiac a on for discovered screens inhibit specific was activator performed than small-molecule the the A rather those motor. on activate of to that performed compounds contrast one been in kinesins, also however, have myosins; screens Small-molecule activator Myosin 2011). oc eeainb ieisadmois17 myosins and kinesins by generation Force nrae itneprstroke per distance Increased nrae ubro toe e nttime unit per strokes following of the number in Increased increased be can motor ways: a by output output mechanical Mechanical increased with Motors 3. Box h icvr fsalmlcl niioso h kinesin the of inhibitors small-molecule of discovery The N N N N N nices ntert fAPrlae sal h rate-limiting the usually release, ADP of rate the in increase An uat aentytbe reported. been such yet stroke; not motor is per have nucleotide distance mutants to the rotation binding increase could motor stalk filament of its energy motor. or or free per the produced arm alter force that the Mutants lever Me and 2011; 2012) al., of et al., (Hallen et angle size step the the increase to expected in increase An ees n Taertsb h oo,rsligi faster in elongated strikingly resulting and motor, assays the motility spindles the by in of gliding rates loop microtubule ATPase a and in residue release conserved a affect central Kinesin-14 that kinesin. for mutants time hydrolysis strokes/unit Ncd ATP of increase number to the expected and is rates cycle, kinesin the in step hudse ih ntemcaimb hc h compound the which myosin by but of mechanism omecamtiv of available, rate the the yet structure to increases on not crystal light is bound shed A mecarbil should myosin 2011). omecamtiv with al., cardiac complexed et myosin. for by (Malik time found mecarbil unit ATP per was of strokes rate of This increased number an the in and result hydrolysis to predicted is stroke, power nices ntelnt ftemoi ee r rkinesin or al., arm et (Stewart lever motors myosin the of the velocity of gliding the length increases the stalk in increase An of rate the in increase An endrcl eosrtdb igemlcl assays. single-molecule by not demonstrated has motor directly force per the been force increase increased to although inferred motor, 2003; been per al., has produced et this Yun 2006); 1996; al., al., et et Endres Uyeda 1993; al., et Chandra 1993; ? P P i P i state. i ees n T yrlssi t bec Mlke al., et (Malik absence its in hydrolysis ATP and release ees ymoi cusa h rniinbtenthe between transition the at occurs myosin by release b sethv eetybe eotdta nraeADP increase that reported been recently have -sheet nvivo in Lue l,2012). al., et (Liu P i ees ytemotor. the by release P i ees,wihtigr h myosin the triggers which release, P i ees and release ´ne ´trey Journal of Cell Science h aewya nmois n rdc oedetailed more a produce and myosins, in as way same the central the whether and eut ntgtmcouuebnig olwdb T binding, motor, ATP the of by stroke followed force-producing the which binding, triggers release, microtubule which ADP tight with begins in from cycle motor results the the kinesins, releases the which For binding, actin. ATP by followed stroke, ofnto,sol eov urnl usadn sus uhas such free of issues, the route outstanding escape currently with the resolve together should relevant information function, studies, obtain to to proteins projected mutant and of Sindelar These characterization 2006; Hirokawa, 2010). A and 8–10 Kikkawa Downing, of 2006; resolutions al., smaller cryo-electron reached et high-resolution much (Hirose currently has from their which come microscopy, with structural could New kinesins domain, motors. motor for the by especially production information, that force in changes involved conformational are key currently regarding the provide information to and expected missing state are kinesin respectively, state, ATP-bound myosin the rigor with between changes compared structural states The these myosins. the of state actin-bound triggered is cycle force-producing and the by myosins myosins, the the between For kinesins. differences important of confirmation Cleland 2011; al., 2011). patients et al., show in (Teerlink et failure humans or function or in cardiac dysfunction disease cardiac trials improves with heart clinical mecarbil initial with omecamtiv of that humans reports in potential Recent have failure. intervention findings a These therapeutic in 3). (Box produced for ways rat it, be different increase noteworthy, potentially of to is than number could of function This motors motor with disrupt ‘improved’ 2011). to dogs although al., easier contractility is that et in it that (Malik function given showed the failure cardiac heart studies improves induced increases functional and cardiomyocytes muscle, mecarbil cardiac omecamtiv by output applications. clinical potential 2012) for al., also the and et understanding mechanism (Liu for implications motor Ncd important new have kinesin-14 would to for 3), (Box recently lead reported such could output, those mechanical as myosin region increase – that this mechanism the changes force-generating mutational of in myosin study the changes Further regarding converter. information residue and lever of helix the relay of effects base the the to near close be by to arm, peptides found was myosin and cardiac an spectrometry, binding labeled as mass derivative identifying The a and 3). using to label mapped (Box affinity expected was motor mecarbil is omecamtiv the it for by site thus an output force, be mechanical producing a in increase and to actin state with predicted interacting binding heads is myosin strong of of effect myosin number overall the cardiac in The increase 1993). on al., force-generating mecarbil et is the (Rayment omecamtiv which – motor arm state, the be lever bound of to the stroke strongly of thought the rotation is enter by It accompanied to 1). myosin (Fig. for state required actin-binding strong and weak nlssta eie h etrso h ih,no-nucleotide ADP tight, weak, the the and of kinesins the features of structural the further state from microtubule-bound defines come force- that to myosin likely analysis and is kinesin mechanism the generating understanding in progress Future Perspectives and Conclusions microtubule. the from motor the of release and 18 h rpriso mcmi eablpoieastriking a provide mecarbil omecamtiv of properties The mechanical increasing in effect proposed its with Consistent P i ees,wihrslsi ih ci idn n h power the and binding actin tight in results which release, ora fCl cec 2 (1) 126 Science Cell of Journal P b i seto ieisdsot rtit in twists or distorts kinesins of -sheet rmtemtratrAPhydrolysis, ATP after motor the from P i release ? P ˚ i nrs .F,Ysik,C,Mlia,R .adVl,R D. R. Vale, and A. R. Milligan, C., Yoshioka, F., N. Endres, L. W. DeLano, ns .P n ors .R. N. Morris, and P. A. Enos, Ebel, A., Blangy, A., D. Skoufias, L., Lebeau, I., Crevel, P., J. Simorre, S., DeBonis, oru,P-. wee,H .adHuus,A. Houdusse, and L. H. Sweeney, P.-D., Coureux, P. S. Gilbert, and M. T. Kapoor, 3rd, E., J. Gatial, C., J. Cochran, oru,P-. el,A . Me L., A. Wells, P.-D., Coureux, V., J. J. Murray, Mc M., E. Nifontov, R., Senior, R., J. Teerlink, F., G. J. Cleland, hi oc-eeaigmcaimi niiae oshow to energy into anticipated enzymes. insight ATP-hydrolyzing further by is lend – transduction will force-producing structure that mechanism overall differences differ unexpected the in force-generating motors myosins dynein unraveling and The their stage. kinesins earlier from which much substantially a at for comparison myosin is for and mechanism interest dyneins, vital kinesin of the be with will by information generation This motors. force of understanding lny .E,Bhk-ak,W . nrasn .O . oefl,S .and S. S. Rosenfeld, L., O. J. Andreasson, M., W. Behnke-Parks, E., B. A. Clancy, S. Endow, and A. Lockhart, P., H. Erickson, D., E. Salmon, R., Chandra, hl u omnaywsbigpeae o ulcto,we publication, for prepared being was Commentary Dimes our of While proof in March added Note the months. Deposited 12 S.A.E.]. and after to release S.A.E.]; for 1-FY07-443 PMC NO. in to number grants to [grant GM046225 by GM097079 Foundation numbers supported and [grant is Health laboratories of F.J.K. Institutes our National in the proteins from motor on Work for Cong Funding crystal Amalia and a publication, of 2. to coordinates Fig. with prior for assistance 4AP0) Kozielski (PDB preprints Frank sending structure publication, for Kozielski to Frank and prior Houdusse Anne thank We Acknowledgements atr .P,Co . i,L n ae .D. R. Vale, and L. Jin, C., Cho, P., A. Carter, and C. F. McCormick, Moores, and A. Z., D. Sanders, Maliga, R., H. B., Bourne, Honig, J., Vendome, M., W. J. Behnke-Parks, B. Schnapp, and B. S. L. Goldstein, M., S. 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