High-resolution structures of kinesin on microtubules provide a basis for nucleotide-gated force-generation
Conventional kinesin is the founding member of a superfamily of molecular motors that use the energy of ATP hydrolysis to transport cargo along microtubules , serving essential roles in a wide variety of cellular processes , most notably mitosis and neuronal transport . More than 20 different members of the kinesin superfamily are identified with mitosis alone , underscoring the motor's fundamental importance in the cellular life cycle ( Rath and Kozielski , 2012 ) . Given the important role of this motor protein in health and disease , and particularly its growing prominence as a therapeutic drug target ( Rath and Kozielski , 2012 ) , it is of considerable interest to understand the basic structural and functional features of this motor in molecular detail . Conventional kinesin ( kinesin-1 ) dimerizes via an extended stalk domain that forms a coiled coil ( Figure 1A ) , so that the two catalytic motor domains are situated at one end of the coiled-coil , while cargo-binding domains are found at the opposite end . During active motility , the dimerized motor domains take alternating , eight nanometer steps toward the microtubule plus end , tracking along single protofilaments ( Gennerich and Vale , 2009 ) . Underlying this behavior , each motor domain cycles between conformations that are strongly attached to the microtubule ( no-nucleotide and ATP-bound ) and ones that are weakly attached ( ADP-bound ) . Additionally , binding of ATP during the microtubule-attached phase of the motor domain causes a structural element called the neck linker ( Figure 1A ) to dock along the side of the motor domain in the plus end direction ( Rice et al . , 1999 ) . The neck linker connects the C-terminus of the motor domain to the stalk , so that docking is accompanied by translocation of the cargo and the partner in the direction of travel . These nucleotide-dependent behaviors are thought to operate together to drive motility and force production . 10 . 7554/eLife . 04686 . 003Figure 1 . Kinesin-microtubule complex at 5–6 Å resolution . ( A ) Schematic of a stepping kinesin dimer on a microtubule protofilament . ( B ) Cross-sections of the reconstructed co-complexes of no-nucleotide kinesin ( left ) and ADP•Al•Fx ( right ) with the microtubule , running parallel to a tubulin protofilament . Fourier Shell Correlation curves and other diagnostic information related to 3D refinement and reconstruction are found in Figure 1—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 04686 . 00310 . 7554/eLife . 04686 . 004Figure 1—figure supplement 1 . Statistics and diagnostic images related to image processing and 3D reconstruction of the kinesin-microtubule complex . ( A ) , ( B ) FSC curves for our 3D reconstructions of microtubules decorated by no-nucleotide and ADP•Al•Fx bound kinesin ( respectively ) . These show signal nearly to the Nyquist frequency , dropping below the 0 . 143 threshold ( 1 ) in the 5–6 Å range . Bold lines correspond to reconstructions in which motion correction was only applied to the initial images . Thin lines reflect a gain of 0 . 5–1 Å in resolution that was achieved by dividing each box segment into three sub-averages of five video frames each , and then separately refining the position and Euler angles of the three sub-averages . ( C ) Representative image of the ADP•Al•Fx sample , after motion correction . ( D ) Post-processed diagnostic image for a 14-protofilament microtubule similar to that shown in ( C ) , depicting the filament in a longitudinally compressed form . Each visible 8 nm repeat ( marked with black arrows ) corresponds to ∼8 repeats in the original filament image that have been averaged together , yielding an 8-fold shortened image with improved signal-to-noise ratio . Note that the contrast has been inverted in this image compared with ( C ) . ( E ) Diffraction pattern obtained by straightening the microtubule imaged in ( D ) using in-plane orientation parameters obtained from the final cycle of reference alignment . Thon rings are visible to beyond 8 Å resolution in all directions . Possibly due to thick ice , Thon rings were rarely if ever observed to extend beyond ∼6 Å resolution , despite the combination of high-voltage instrument and direct electron detectors used in the current work . ( F ) Example of alpha-helical density from tubulin where helical pitch ( arrows ) can be resolved . ( G ) Example of beta-sheet density where individual strands are partially resolved ( arrows ) . DOI: http://dx . doi . org/10 . 7554/eLife . 04686 . 004 The key structural transitions that underlie kinesin's stepping behavior remain uncertain . Kinesin possesses a Walker-type active site architecture , in which one important structural element ( the P-loop ) coordinates the nucleotide alpha- and beta-phosphates , while two ‘switch’ loops serve as nucleotide response elements ( Kull et al . , 1996; Sablin et al . , 1996 ) . It has long been suspected that these switch loops act as ‘gamma phosphate sensors’ , transitioning from ‘open’ to ‘closed’ conformations in response to ATP . In this type of scheme , closure of the switch loops would propagate across the motor domain via a series of linked allosteric rearrangements in order to dock the neck linker to the motor domain . Moreover , because the switch II loop is N-terminally adjacent to one of kinesin's microtubule-interacting subdomains , sometimes called the switch II cluster , it was further proposed that kinesin could use the same gamma phosphate-sensing mechanism in order to control its microtubule affinity ( Vale and Milligan , 2000; Kikkawa et al . , 2001 ) . Owing to more recent structural findings , however , the preceding ideas are increasingly regarded as incomplete or even incorrect ( Sindelar , 2011 ) . A major barrier to further progress in elucidating kinesin's mechanism has been the inability of cryo-EM methods to much exceed nanometer resolution with kinesin-microtubule complexes ( Sindelar , 2011 ) , making it difficult or impossible to determine which crystal structures of kinesin ( if any ) correspond to the motor's functionally relevant , microtubule-attached states . Recently , a series of X-ray crystal structures of kinesin have been solved in the presence of ATP analogs that are thought to closely resemble kinesin's ATP state on microtubules; these include not only two structures of kinesin solved in the absence of protein co-factors ( Parke et al . , 2010; Chang et al . , 2013 ) , but also a structure of kinesin co-complexed with a dimer of tubulin ( Gigant et al . , 2013 ) . However , it has remained unclear how closely these structures resemble the corresponding state of kinesin when bound to its physiological substrate ( an intact microtubule ) , and structure models for other nucleotide states of microtubule-attached kinesin have remained elusive . These issues have left open the possibility that one or more of kinesin's vital functions may be governed by allosteric pathways not envisioned in the gamma phosphate sensor scheme , possibly involving twisting of the motor's central beta sheet ( Hirose et al . , 2006; Kull and Endow , 2013; Arora et al . , 2014 ) . Of particular importance is whether switch loop closure is triggered by ATP binding ( as in the gamma phosphate sensor scheme ) or by some other event , which would allow ATP binding to have a distinct function . The coordination that occurs between two heads in a kinesin dimer is even more challenging to explain with existing structural models . Recent single-molecule optical trapping studies of kinesin dimers are most consistent with a model in which the leading head of a stepping dimer has substantially reduced nucleotide affinity compared with the trailing head ( Guydosh and Block , 2006; Clancy et al . , 2011 ) . Related to this observation , it was also shown that the nucleotide affinity of the kinesin motor domain depends on the direction of a load externally applied to the neck linker ( Uemura and Ishiwata , 2003 ) . These lines of evidence indicate that kinesin's neck linker may serve as a control lever , ‘gating’ the affinity for nucleotide depending on whether it points forward ( as in a trailing head ) or backward ( as in a leading head ) ( Asenjo et al . , 2003; Gennerich and Vale , 2009 ) . However , the precise nature of this and other proposed gating functions remains under debate ( Shastry and Hancock , 2010 ) and , similar to the monomer mechanism itself , there is little consensus on the structural basis of any type of gating mechanism in dimeric kinesin ( Sindelar , 2011 ) . To address these questions , we exploited recent advances in cryo-EM methodology to obtain structure models of the kinesin motor domain complexed with microtubules in the absence of nucleotide and following binding of the non-hydrolyzable transition state analog , ADP•Al•Fx . Analysis of these structures leads us to infer that kinesin's switch loops , controlled by microtubules , serve as a nucleotide exchange factor while ATP binding docks the neck linker via a clamshell distortion of the active-site nucleotide cleft . The corresponding allosteric pathways deduced from our structures are distinct from prior proposals that originated from X-ray structures of free kinesin or from lower-resolution cryo-EM reconstructions , including a very recent study where the reported resolution was in the ∼6–7 Å range ( Atherton et al . , 2014 ) . Moreover , the resulting mechanistic model for the kinesin monomer can be used to explain key aspects of ‘gating’ between heads of a stepping kinesin dimer and provides a paradigm for future study of other motor proteins such as dynein and myosin , where atomic structures of the motor-filament complex have not yet been determined .
In order to clarify the mechanism of kinesin's microtubule-attached cycle , we solved high-resolution cryo-EM reconstructions of the human monomeric kinesin motor domain ( K349 ) attached to the microtubule in two different chemical states ( Figure 1B ) , representing the motor before and after ATP binding ( no-nucleotide and in the presence of ADP•aluminum fluoride ( ADP•Al•Fx ) ) . Through the application of recent advances in instrumentation and image processing , including the use of a high-speed electron-counting detector and algorithms to correct for beam-induced sample motion ( Li et al . , 2013 ) ( See ‘Materials and methods’ ) , we were able to obtain a resolution of ∼5–6 Å in both of these maps ( Figure 1—figure supplement 1A , B ) , a significant increase over previous studies of the kinesin-microtubule complex ( Sindelar , 2011; Atherton et al . , 2014; Goulet et al . , 2014 ) . In some regions of the maps , the pitch of α-helices was resolved as were individual beta strands , indicating a resolution of at least 5 Å ( Figure 1—figure supplement 1F , G ) . Features in the kinesin region of the map , however , appear to be limited to ∼6 Å resolution , probably due to distortions of the helical lattice that could not be corrected by our image-processing algorithms . Hence , we filtered our maps to remove all signal beyond 6 Å resolution before proceeding with the analysis described below . Even after this filtering , both of our cryo-EM maps resolved all beta sheets , alpha helices , and many loops in both kinesin and tubulin as discrete entities . In particular , the paths of the motor's three principal microtubule-binding loops ( L8 , L11 , L12 ) were directly resolved ( Figure 2 ) , which substantially aided further analysis . 10 . 7554/eLife . 04686 . 005Figure 2 . Clamshell-like closure of kinesin's nucleotide cleft triggered by ATP binding on microtubules , as revealed by cryo-EM density maps of no-nucleotide kinesin ( top panels ) and the ADP•Al•Fx state ( bottom panels ) . Nucleotide cleft closure is coupled , via rotation of the N-terminal subdomain , to opening of a ‘docking cleft’ on the opposite side of the motor domain and accompanying docking of the neck linker ( A ) , ( B ) , ( D ) , ( E ) two different slices of density perpendicular to the microtubule axis , detailing the N-terminal and upper domains ( respectively ) . Microtubule contacts are denoted by colored arrowheads . ( C ) , ( F ) Side view of motor domain facing the nucleotide active site . Thin colored cylinders in panels D–F depict rotational axes that describe re-orientations of the N-terminal domain ( blue ) and upper domain ( magenta ) compared with the no-nucleotide state . Atomic models superposed on these maps in this and the following figures were derived from our cryo-EM data by molecular dynamics flexible fitting simulations as described in the text ( see Videos 1–3 and Figure 2—figure supplements 1–3 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 04686 . 00510 . 7554/eLife . 04686 . 006Figure 2—figure supplement 1 . Assessing the convergence of the MDFF simulations . ( A–B ) , Backbone RMSD values as a function of time , measured separately for tubulin and kinesin against the starting model , for MDFF simulations of the no-nucleotide and ATP states of kinesin ( respectively ) . The bold black line indicates the corresponding value of the EM potential scaling factor ( ξ ) , which follows a linear ramp function ( See ‘Materials and methods’ ) . Time points selected to represent the fitted complex ( with minimal over-refinement artifacts ) are indicated by dashed lines . ( C–D ) , Backbone RMSD values in follow-up simulations starting with the selected structures in ( A ) and ( B ) , respectively , where the cryo-EM potential was turned off but the coordinates of tubulin were restrained in the ‘straight’ conformation ( see ‘Materials and methods’ ) . DOI: http://dx . doi . org/10 . 7554/eLife . 04686 . 00610 . 7554/eLife . 04686 . 007Figure 2—figure supplement 2 . Improved interactions at the kinesin-tubulin interface following initial equilibration of the 4HNA starting model ( see ‘Materials and methods’ ) . ( A ) Overview of the kinesin-microtubule complex , highlighting the region selected for the close-ups in panels ( B ) – ( D ) . Four charged residues from kinesin's microtubule binding regions are labeled . The hydrogen-bonding geometry for R278 ( loop L12 ) is extremely poor and R161 has no interaction partners in the 4HNA starting model ( B ) , likely due to resolution limitations and anisotropy in the X-ray data . In both the no-nucleotide and ATP simulations ( C and D , respectively ) , these residues stably interact with complementary charge partners in tubulin and/or adjacent residues of kinesin . DOI: http://dx . doi . org/10 . 7554/eLife . 04686 . 00710 . 7554/eLife . 04686 . 008Figure 2—figure supplement 3 . Mobility of loop L9 ( corresponding to the switch I loop ) increases in simulations of unrestrained , no-nucleotide kinesin bound to microtubules , compared with simulations of the ATP state . These loop motions account for differences in cryo-EM density features for this loop seen in Figure 2C , F . These simulations were restarted from the endpoint of the corresponding MDFF simulations , but with the cryo-EM energy term turned off ( see ‘Materials and methods’ ) . The kinesin structure in these simulations exhibits substantially larger conformational fluctuations when compared to the MDFF simulations ( not shown ) , indicating that addition of the cryo-EM energy term substantially restricts breathing movements that ordinarily occur at the simulated temperature ( 300 K ) . ( A ) Four representative conformations in the trajectory of unrestrained no-nucleotide kinesin , revealing that loop L9 can flap away from the nucleotide pocket when the gamma phosphate is absent . ( B ) Four representative conformations from the ATP-bound kinesin trajectory , reflecting the fact that L9 is stably engaged with the gamma-phosphate and is unable to fluctuate greatly . DOI: http://dx . doi . org/10 . 7554/eLife . 04686 . 008 Comparison of our no-nucleotide and ATP-state maps reveals that binding of the ATP analog causes an articulated conformational change in the kinesin domain that involves the concerted motion of three relatively well-defined subdomains ( Figure 2 ) . Two of these subdomains result from dividing the central beta sheet at the junction between the beta strands anchoring the P-loop and the switch II loop ( beta-3 and beta-7 , respectively ) . The third subdomain , sometimes called the switch II cluster , contains kinesin's principle microtubule interaction elements including the switch II helix and C-terminally adjacent loop L12 ( Woehlke et al . , 1997 ) . Because these three subdomains are closely analogous to subdomains that were previously identified in the myosin motor protein , known as the upper 50kD , lower 50kD , and N-terminal subdomains ( Sweeney and Houdusse , 2010 ) , we have adopted a similar naming convention in current work . Consistent with earlier cryo-EM observations ( Kikkawa et al . , 2001; Kikkawa and Hirokawa , 2006; Sindelar and Downing , 2010 ) , the lower subdomain remains largely fixed on the microtubule surface in our maps ( rotation of less than 5° ) . In contrast , the N-terminal subdomain makes a ∼22° seesaw-like motion atop the lower subdomain ( compare Figure 2A , B to Figure 2D , E ) in response to binding of the ATP . This action carries the neck linker attachment site ( at the C-terminus of helix alpha 6 ) up and away from the lower subdomain , concomitantly forming a cavity and delivering the neck linker into it . Correspondingly , in the ATP analog state , we observed density consistent with a docked conformation of the neck linker , extending towards the microtubule plus end ( Figure 1B; Figure 2A , B ) , while in the no-nucleotide state the neck linker was evidently disordered ( Figure 1B; Figure 2D , E ) , consistent with previous observations ( Rice et al . , 1999; Sindelar and Downing , 2007 , 2010 ) . Thus , seesaw movement by the N-terminal subdomain couples ATP binding to docking of the neck linker , in agreement with the conclusions of several previous cryo-EM studies that visualized nucleotide-induced transitions of kinesin at lower resolution ( Kikkawa and Hirokawa , 2006; Sindelar and Downing , 2007 , 2010; Peters et al . , 2010; Goulet et al . , 2014 ) . In contrast to previous analyses of these structural states of kinesin , however , our data reveal that the seesaw movement identified for the N-terminal subdomain does not extend to the other half of the beta sheet ( the upper subdomain ) . Rigid-body fitting experiments using fragments of kinesin X-ray structures indicate that the upper subdomain rotates by much less ( ∼11° ) than the N-terminal subdomain ( Figure 2D , E ) . Moreover , these fitting experiments indicate that the upper subdomain rotates about an axis that runs directly through the active site , thus predicting that the switch loops ( which are anchored in the upper subdomain ) would remain largely fixed in space proximal to the switch II helix as the upper subdomain rotates ( Figure 2E , F ) . Related to this observation , we discovered a striking resemblance in the conformation of the switch II loop before and after binding of the ATP analog ( Figure 2C , F; Figure 3 ) . Both of our maps exhibit a contact between the switch II loop and switch II helix , coinciding with a pair of hydrogen bonds formed between the backbone of E236 and the side chain of N255 ( respectively ) in X-ray structures of kinesin's ATP analog-bound state ( Parke et al . , 2010; Chang et al . , 2013; Gigant et al . , 2013 ) ( Figure 3A , B ) . Proximal to this contact , our maps resolve another contact between the switch II helix and the microtubule surface , corresponding to a hydrogen bond between N255 and M413 of alpha tubulin in the kinesin–tubulin co-complex structure ( Gigant et al . , 2013 ) . Thus , our maps indicate that the upper and lower domains are pinned down against the microtubule surface by a three-way interaction between the switch II loop , the switch II helix and alpha tubulin during the ATP binding transition . 10 . 7554/eLife . 04686 . 009Figure 3 . The effect of nucleotide state and microtubule attachment on kinesin’s ‘nucleotide’ and ‘polymer’ clefts . Shown is an enlarged , cutaway view of the active site ( rectangular region indicated in Figure 2C , F ) . Density features that define the nucleotide cleft , corresponding to the switch II loop and the P-loop , are indicated by ‘SwII’ and ‘P’ ( respectively ) in panel A . Hollow and solid wedges denote open and closed states ( respectively ) of the nucleotide and polymer clefts . Panels A–B show isosurface renderings our cryo-EM maps , while panels C–D show synthetic density maps generated from X-ray structures of our identical kinesin construct in its ADP-bound state , filtered to a similar resolution ( 6 Å ) as the experimental maps in panels A–B . Figure 3—figure supplemental 1 shows the corresponding features for synthetic 6 Å-resolution maps generated from our atomic models , as well as for two additional ADP-bound crystal structures of kinesin . DOI: http://dx . doi . org/10 . 7554/eLife . 04686 . 00910 . 7554/eLife . 04686 . 010Figure 3—figure supplement 1 . Visualizing nucleotide and polymer clefts in synthetically rendered maps of kinesin at 6 Å resolution . The view is the same as in Figure 3 . Panels ( A–B ) were generated from the final atomic models for these maps generated by our MDFF simulation protocol , while panels ( C–D ) were rendered from the listed PDB coordinates . DOI: http://dx . doi . org/10 . 7554/eLife . 04686 . 010 A striking difference between our cryo-EM maps , however , is a distinct upward displacement of the P-loop away from the switch II loop in our no-nucleotide map . This movement appears to be a direct consequence of the 22° rotation made by the N-terminal subdomain , to which the P-loop is anchored; the rotation translates the P-loop site ∼4 Å away from the microtubule surface . Consistent with this interpretation , while the P-loop forms a visible contact with the switch II loop in our ATP analog map ( Figure 3B ) , a gap appears between these elements in the no-nucleotide map ( Figure 3A ) . In contrast , the P-loop and switch II loop maintain very close contact in ADP-bound X-ray structures of kinesin ( Figure 3C , D and Figure 3—figure supplement 1C , D ) . At the same time , however , the switch II loop fails to make close contact with the switch II helix in the ADP-bound X-ray structures ( Figure 3C , D and Figure 3—figure supplement 1C , D ) . Thus , kinesin's tightly microtubule-attached states ( Figure 3 , top panels ) are distinguished from the weakly attaching , ADP-bound states ( Figure 3 , bottom panels ) by the presence of a gap between the switch II helix and the switch II loop; correspondingly , we will refer to this feature as the ‘polymer cleft’ . Similarly , the presence of a gap between the P-loop and the switch II loop correlates with the absence of bound nucleotide; we will therefore refer to this latter feature as the ‘nucleotide cleft’ . In the microtubule-attached states captured by our cryo-EM maps , the closed polymer cleft holds the upper and lower subdomains together while ATP binding initiates a clamshell-like action between the N-terminal domain and upper/lower subdomain assembly to close the nucleotide cleft . We used the MDFF package ( Trabuco et al . , 2008 ) to develop an atomic model for our ATP analog map , starting with the crystal structure of the same chemical state of the same kinesin construct co-complexed with a tubulin dimer ( Gigant et al . , 2013 ) . This calculation employed an all-atom representation with an explicit solvation model , combined with a novel , customized steering potential derived from our cryo-EM maps ( See ‘Materials and methods’ and Figure 2—figure supplement 1 ) . In the resulting structure model , the principle conformational change observed is a straightening of the tubulin subunits from the conformation seen in 4HNA in order to accommodate the microtubule lattice ( Video 1 ) . Despite this change in tubulin , the conformation of kinesin is well conserved ( 1 . 08 Å backbone RMSD with the 4HNA structure for residues 9–333 ) . Some differences with the 4HNA structure are evident at the kinesin-microtubule interface of our simulations ( Figure 2—figure supplement 2 ) , but these involve non-conserved side chains that re-orient to form more favorable contacts during the equilibration phase of the simulation ( where the backbone conformations of kinesin and tubulin were placed under a strong harmonic constraint , prior to application of the EM steering potential ) . The MDFF calculation thus indicates that the conformation of kinesin visualized in the 4HNA structure is largely conserved when tubulin is incorporated into the microtubule lattice . 10 . 7554/eLife . 04686 . 011Video 1 . Animation depicting the conformational transition observed in our MDFF simulation of the ATP bound kinesin–microtubule complex . A ribbon diagram of the molecular structure is superimposed on a semitransparent isosurface of the EM density map , filtered to 6 Å resolution . The video runs from the beginning of the MDFF simulation up to t = 1 . 2 ns ( corresponding to the selected final model ) . The second half of the video plays the same transition backward . Coordinates were extracted at 50 ps intervals from the simulation trajectory . To aid in viewing , the depicted coordinate trajectory was smoothed using a 5-frame sliding window , using the ‘smoothmd . py’ script available from the UCSF Chimera web site ( http://plato . cgl . ucsf . edu/trac/chimera/attachment/wiki/Scripts/smoothMD . py ) . DOI: http://dx . doi . org/10 . 7554/eLife . 04686 . 011 We repeated the above MDFF procedure to obtain a structure model for the no-nucleotide cryo-EM map . To match the chemical and structural features identified in this latter map , we removed ATP and magnesium from the active site of the 4HNA structure and deleted residues 321–349 of the neck linker , for which density in the map was weak or absent . In effect , this simulation protocol is designed to reverse the isomerization induced by ATP binding in microtubule-attached kinesin , yielding a detailed prediction for the conformation of kinesin in the absence of nucleotide ( Video 2 ) . 10 . 7554/eLife . 04686 . 012Video 2 . Animation depicting our MDFF simulation for no-nucleotide kinesin , up to t = 1 . 4 ns . DOI: http://dx . doi . org/10 . 7554/eLife . 04686 . 012 The atomic models derived by the above procedure recapitulate the three-subdomain articulated movement identified by our rigid-body fitting analysis , and moreover , provide a detailed molecular explanation for the observed features at the active site . Remarkably , the switch loops remain ‘closed’ in an identical hydrogen-bonding network in our structural models for both nucleotide states . The hydrogen bond partners in this network , which are shared by the 4HNA starting model , extend beyond the switch loops and include not only a universally conserved salt bridge between R203 ( switch I ) and E236 ( switch II ) , but also several universally conserved residues in the switch II helix ( E250 , N255 ) and L7 from the upper subdomain ( Y138 ) ( Figure 4 , middle and lower panels ) . These interactions thus lock the upper and lower subdomains together , defining the closed state of the polymer cleft; the same network also locks the lower domain against the surface of alpha tubulin via a conserved hydrogen bond between N255 and the backbone of M413 from alpha tubulin . 10 . 7554/eLife . 04686 . 013Figure 4 . Atomic models capturing open and closed states of kinesin's nucleotide and polymer clefts for principle structural intermediates in the kinesin cycle . No-nucleotide and ATP-bound models of the kinesin-microtubule complex ( this work ) are depicted in panels A , D , G and B , E , H ( respectively ) . A pair of X-ray crystal structures of the ADP-bound kinesin K349 construct ( PDB ID: 1BG2 , 1MKJ , which have undocked and docked neck linkers , respectively ) are superposed in C , F and I . Panels G–I show rotated views of the structures , presenting the view from the microtubule interior looking outward at the kinesin interface . Universally conserved residues involved in the depicted structural transitions are rendered as ball and stick figures , and hydrogen bonds are depicted as solid green lines . Hollow and solid wedges denote open and closed states ( respectively ) of the nucleotide cleft and the polymer cleft , while the star in panels F and I indicates the site of predicted steric clashes between the switch II helix and alpha tubulin . For comparison , views of the active sites of the F1-ATPase and myosin motors in no-nucleotide and ATP analog-bound states , corresponding to panels A and B , are shown in Figure 4—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 04686 . 01310 . 7554/eLife . 04686 . 014Figure 4—figure supplement 1 . Nucleotide cleft closure induced by ATP analogs in the myosin and F1-ATPase motor proteins ( compare with Figure 4A–C ) . Changes in the hydrogen-bonding pattern between P-loop and switch II residues between in these motors closely match the changes predicted by our molecular dynamics models of kinesin's no-nucleotide and ATP states . Both of these other motors also undergo substantial distortions in the supporting beta-sheet conformation that accompany the P-loop movement . ( A–B ) Active site of the subunit of bovine mitochondrial F1-ATPase in no-nucleotide and ADP•Al•Fx states , respectively , from a crystal structure of the inhibited ( αβ ) 3-γ co-complex ( PDB ID: 1H8E ) c-d . Active site of the myosin V motor domain from X-ray structures solved in the absence and presence of ADP•Al•Bex ( PDB ID's 1W8J and 1W7J , respectively ) . Viewing orientations of the structure pairs in top and bottom panels are generated by aligning the backbone atoms in the beta-strand residues N-terminal to the switch II aspartic acid ( D256 and D437 , respectively ) . DOI: http://dx . doi . org/10 . 7554/eLife . 04686 . 014 While switch loops are thus stably maintained in a closed arrangement in these structure models , we observed a functionally significant rearrangement of the P-loop that accompanies opening of the nucleotide cleft , for the nucleotide-free model . As the P-loop pulls away from the switch loops , a bifurcated hydrogen bond breaks up between T92 at the base of the P-loop and S202/D231 in switches I and II ( Figure 4A , B and Video 3 ) . This action substantially perturbs the magnesium binding site , which is primarily formed by T92 . A second residue in the P-loop affected by its translation away from the switch loops is K91 , which normally coordinates the nucleotide alpha- and beta-phosphate groups . In the nucleotide-free structure , K91 breaks two hydrogen bonds with a pair of backbone carbonyl oxygens within the interior of the P-loop and swivels around to interact with D231 in switch II , replacing the hydrogen bond this latter residue formerly made with T92 . In doing so , K91 loses its ability to coordinate the nucleotide alpha- and beta-phosphates . The resulting side chain configuration within the active site of our nucleotide-free kinesin model is strikingly similar to what is seen in X-ray structures of nucleotide-free F1-ATPase and myosin motors , which possess homologous active-site architectures ( Figure 4—figure supplement 1 ) . Thus , opening of the nucleotide cleft in our no-nucleotide model specifically disrupts the coordination of both nucleotide phosphate and magnesium within the P-loop . 10 . 7554/eLife . 04686 . 015Video 3 . Alternative depiction of the transition presented in Video 2 , detailing the side chains and hydrogen bonds that compose the closed switch loop network ( Y138 , R203 , E236 , E250 , N255 ) , as well as residues P-loop residues K91 , T92 , and their interacting partner D231 from the switch II loop ( compare with Figure 3 ) . Hydrogen bonds formed at the beginning and endpoints of the simulation are depicted with green lines . Note that in this video the position and orientation of the outer surface of tubulin ( helices H11-H12 ) is held fixed . In order to facilitate looping presentations of the video , the second half of the video presents the same frames of the MDFF trajectory but in reverse order . DOI: http://dx . doi . org/10 . 7554/eLife . 04686 . 015 In order to identify the basis for microtubule-affinity regulation of kinesin by ADP and vice versa , we compared our structures of microtubule-attached kinesin with two X-ray structures ( PDB ID's 1MKJ , 1BG2 ) of the same construct in an ADP-bound , weakly attaching state ( Kull et al . , 1996; Sindelar et al . , 2002 ) . The relative arrangements of the upper and lower domains in the 1BG2 and 1MKJ structures , corresponding to undocked and docked states of the neck linker , closely align with our no-nucleotide and ATP state kinesin models ( respectively ) . We discovered , however , that both the 1BG2 and 1MKJ structures are prohibited from assuming favorable binding orientations on the microtubule surface due to the conformation of residue N255 . As illustrated in Figure 4D–F , the N-terminal coils of the switch II helix are unwound in both ADP-bound structures . Consequently , the peptide backbone at residue N255 would point directly at the H11–H12 loop of alpha tubulin , generating a substantial steric overlap ( Figure 4F , I ) . Thus , the 1BG2 and 1MKJ conformations appear incapable of strong microtubule attachment , consistent with the weak experimentally observed microtubule affinity of kinesin's ADP state ( Rosenfeld et al . , 1996 ) . Further comparison of our structure models to other atomic structures of ADP-bound kinesin ( Figure 5 ) consistently demonstrates a clash between alpha tubulin and residue N255 ( and/or adjacent residues in the switch II helix ) , for the ADP-state structures . While ADP-state crystal structures of kinesin are largely similar in their active-site features to the 1BG2 and 1MKJ structures , some of these exhibit an alpha-helical conformation for N255 and adjacent residues that N-terminally extends the switch II helix . However , as illustrated in Figure 5B , when these latter conformations of kinesin are placed in a tight-binding orientation on the microtubule ( as defined by our no-nucleotide or ATP state models ) , the N-terminus of the switch II helix projects downward into the microtubule surface , introducing a clash between N255 and the H11-H12 loop of alpha tubulin . A recently solved ‘near-rigor’ conformation of the KIF14 motor ( PDB 4OZQ ) ( Arora et al . , 2014 ) , found in a state that binds ADP but not magnesium , partially repairs the clash in Figure 5B by re-orienting the switch II helix to nearly the identical position ( relative to the upper domain ) as seen in our no-nucleotide model ( Figure 5C ) . This reorientation of the switch II helix is accompanied by inward movement of the universally conserved R591 side chain ( equivalent to R190 in our construct ) from loop L7 , apparently dislodging S489 from D638 ( equivalent to T92 and D231 , respectively ) to disrupt magnesium coordination ( Arora et al . , 2014 ) , thus partially mimicking changes observed at the active site of our no-nucleotide structure . However , the side chain of N662 in the 4OZQ structure ( equivalent to N255 ) projects downward toward the microtubule surface , introducing a steric clash with alpha tubulin . 10 . 7554/eLife . 04686 . 016Figure 5 . Putative intermediate states that lead to opening of kinesin’s nucleotide cleft , accompanied by ADP release , upon microtubule attachment . ( A ) X-ray structure of Mg•ADP-bound K349 kinesin ( PDB ID 1BG2 ) . ( B ) X-ray structure of Mg•ADP-bound KIF1A kinesin ( PDB ID: 1I5S ) . ( C ) X-ray structure of ‘near rigor’ , ADP-bound KIF14 kinesin ( PDB ID: 4OZQ ) ( D ) No-nucleotide kinesin-microtubule complex ( this work ) . Structures in panels A–C are aligned with the no-nucleotide K349 model in panel D by strand b7 of the central beta sheet , which connects to the switch II loop; this alignment approximately maintains the orientation of the upper subdomain , thus preserving a contact between L8 within this subdomain and alpha tubulin ( see Figure 2 ) . In panels A–C , predicted steric clashes between alpha tubulin and residues corresponding to I254 and/or N255 ( K349 numbering ) are marked with a red star . DOI: http://dx . doi . org/10 . 7554/eLife . 04686 . 016 Further comparison of the 4OZQ structure to our no-nucleotide model ( Figure 5D ) reveals that , while the overall arrangement of the N-terminal , upper , and lower subdomains is nearly identical , reorientation of N255 in the no-nucleotide structure is accompanied by striking shift of the switch II loop toward this residue and away from the P-loop . This shift opens the nucleotide cleft and closes the polymer cleft ( See also Figure 3—figure supplement 1D ) and allows the switch II loop to assume its closed conformation that simultaneously interacts with N255 as well as R203 from switch I . The conformation of the N255 side chain that accompanies closure of the switch II loop is highly constrained by a pair of hydrogen bonds to the backbone of E236 , aligning N255 in order to interact with M413 of alpha tubulin ( Figure 4G , H ) . Thus , closure of the polymer cleft in our no-nucleotide model appears to be closely linked to favorable microtubule interactions by N255 . Conversely , as indicated in Figure 4F , I and Figure 5A–C , the open arrangement of the polymer cleft in kinesin's ADP states is evidently inconsistent with tight microtubule attachment , owing to steric interference of N255 and neighboring residues . Previously , it has been speculated that release of nucleotide in kinesin may be linked to twisting of the central beta sheet ( Gigant et al . , 2013; Kull and Endow , 2013; Arora et al . , 2014 ) . In contrast to this expectation , however , we discovered that the degree of end-to-end twisting in both our no-nucleotide model as well as the previously reported ‘rigor-like’ ADP complex of KIF14 falls within the range observed in other X-ray structures of ADP-bound kinesin ( Videos 4–6 ) . However , we identified another mode of distortion in our kinesin models , analogous to the action of an archery bow ( Figure 6 and Videos 7 , 8 ) , that would couple strong nucleotide binding to elastic strain in the peptide backbone . In the conformation , we identify as ‘relaxed’ , corresponding to the no-nucleotide state , the P-loop withdraws from the switch II loop to open the nucleotide cleft . In order for the P-loop to move back toward switch II and close the nucleotide cleft , the N-terminus of alpha 2 ( to which the P-loop attaches ) must move with it . However , because the C-terminus of alpha 2 is covalently connected to the opposite end of the beta sheet , movement of the P-loop therefore stretches alpha 2 across the motor domain , simultaneously deforming the plus end tip of the beta sheet ( Figure 6A , B , D , E , Figure 6—figure supplement 1A , B and Videos 7 and 8 ) as well as the path of alpha 2 itself ( Figure 6C ) . 10 . 7554/eLife . 04686 . 017Video 4 . Comparison of beta-sheet twist in our no-nucleotide model ( cyan ) and five representative X-ray structures of ADP-bound kinesin ( pale magenta/pale blue ) . Structures in this and all subsequent videos are aligned by residues 226–231 of our kinesin model , as in Figure 6A . The video cycles sequentially through the ADP structures in order of increasing twist: frame 1 , ADP-bound K349 ( PDB ID:1MKJ ) ; frame 2 , ADP-bound KIF1A ( PDB ID:1I5S ) ; frame 3 , ADP-bound K349 ( 1BG2 ) ; frame 4 , ADP-bound KAR3 ( PDB ID:1F9T ) ; frame 5 , ADP-bound Eg5 ( PDB ID:1II6 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 04686 . 01710 . 7554/eLife . 04686 . 018Video 5 . Similar to Video 4 , but substituting our no-nucleotide model with the X-ray structure of ‘rigor-like’ kinesin , in green ( PDB ID: 4OZQ ) . DOI: http://dx . doi . org/10 . 7554/eLife . 04686 . 01810 . 7554/eLife . 04686 . 019Video 6 . Morphing animation illustrating minimal beta-sheet distortion in a comparison of ADP-bound structures of KIF1A ( PDB ID:1I5S ) and ‘rigor-like’ KIF14 ( lacking magnesium; PDB ID:4OZQ ) . The starting structure is that of KIF14 . The hydrogen bond network between residues T87 and E236 ( K349 numbering ) , which defines a closed state of the nucleotide cleft , is depicted and the involved side chains rendered as stick figures . DOI: http://dx . doi . org/10 . 7554/eLife . 04686 . 01910 . 7554/eLife . 04686 . 020Figure 6 . Evidence that nucleotide binding introduces internal strain in kinesin's central beta sheet and helix alpha 2 , mediated by interactions between the P-loop and the switch II loop . Similar comparisons of additional structural states of kinesin are shown in Figure 6—figure supplement 1 and Videos 4–8 . ( A ) Flexing of the beta sheet coupled with motion of alpha 2 in comparisons of no-nucleotide and ATP-bound conformations of microtubule-attached kinesin ( this work ) . Structures are aligned by central region of the beta strand adjoining the switch II loop ( residues 226–231 ) . ( B ) Comparison of no-nucleotide and ADP-bound ( 1MKJ ) structures of kinesin , aligned as in panel A . ( C ) Comparison of no-nucleotide ( current work ) , ADP-bound ( 1BG2 and 1MKJ ) , and ATP-bound ( current work ) states of the K349 kinesin construct aligned by their P-loops , revealing nucleotide-dependent flexing of helix alpha 2 . ( D ) , ( E ) Rotated views of panel A and B , respectively , highlighting distortion in the beta sheet proximal to the P-loop . ( F ) Cartoon depicting the inferred energy storage mechanism , by analogy to stringing an archery bow . The strain appears to be magnified by a cluster of conserved side chains ( I107 , I108 , L229 , I81; see panel A ) that separate alpha 2 and the beta sheet midway along the interface between these elements . DOI: http://dx . doi . org/10 . 7554/eLife . 04686 . 02010 . 7554/eLife . 04686 . 021Figure 6—figure supplement 1 . Relationship between nucleotide state and strain in the polypeptide backbone for a diverse array of kinesin X-ray structures . ( A ) , ( B ) Comparison between our no-nucleotide K349 structure and a ‘rigor-like’ ( ADP-bound ) conformation of KIF14 ( PDB ID: 4OZQ ) . Compare with Figure 6A , B , D , E . ( C–F ) Comparison of the orientation of helix α2 relative to the P-loop , for various crystalized states of kinesin . All alignments in these panels are performed relative to the backbone atoms of residues 85–92 ( the P-loop ) in our no-nucleotide structure ( shown in ( C ) ) . The 4OZQ structure is shown in ( D ) . In ( E ) , five representative structures of ADP•Mg-complexed kinesin are superposed ( PDB ID's: 1BG2 , 1MKJ , 1I5S , 1II6 , 1F9V ) ; in ( F ) , our ATP state model is depicted along with the three currently available X-ray structures of kinesin in which the switch loops are fully closed ( all co-bound with ATP analogs ) ( PDB ID's: 3HQD , 3ZFC , 4HNA ) . DOI: http://dx . doi . org/10 . 7554/eLife . 04686 . 02110 . 7554/eLife . 04686 . 022Video 7 . Comparison of backbone distortions in the central beta sheet and α2 for our no-nucleotide and ATP-bound models of K349 kinesin , illustrated by a morphing animation . Compare with Figure 6A , D . DOI: http://dx . doi . org/10 . 7554/eLife . 04686 . 02210 . 7554/eLife . 04686 . 023Video 8 . Similar to Video 7 , but substituting the X-ray structure of ADP-bound kinesin ( PDB ID: 1MKJ ) for the ATP-bound model . Compare with Figure 6B , E . DOI: http://dx . doi . org/10 . 7554/eLife . 04686 . 023 Comparison of kinesin's active site in various nucleotide states indicates that there are two ways to keep the nucleotide cleft closed , depending on the conformation of the switch loops . If the switch loops are open , corresponding to kinesin's detached ADP states , the switch II loop and the P-loop can interact closely with each other , which would secure the closed conformation of the nucleotide cleft ( Figure 4F ) . Alternatively , when the switch loops are closed , closure of the nucleotide cleft is mainly supported by the gamma phosphate group of ATP itself , which introduces a bridge between the P-loop and the switch II loop that would hold these elements together ( Figure 4E ) . In this way , our structure models indicate that tight ADP binding would be an inherent property of detached kinesin , where the switch loops are free to adopt an open conformation . However , closure of the switch loops ( as accompanies microtubule attachment in our structure models ) eliminates the ability of switch II to latch onto the P-loop , which would thus allow the nucleotide cleft to relax into its open conformation unless gamma phosphate is present . The preceding analysis has indicated that residue N255 in kinesin plays a key role in the motor's microtubule response pathway . In effect , this site appears to act as a ‘linchpin’ that links closure of kinesin's switch loops to interactions at a single site on alpha tubulin ( See Figure 4D–F ) . A lysine point mutation at the N255 site was previously shown to eliminate coupling between microtubule attachment and motor nucleotide state , allowing kinesin to bind tightly to the microtubule in all nucleotide states ( Song and Endow , 1998; Yun et al . , 2001; Auerbach and Johnson , 2005 ) . Moreover , the microtubule-stimulated ADP release rate for this point mutant is severely attenuated , falling to only slightly above the basal level ( Auerbach and Johnson , 2005 ) ( Figure 7A , B ) . Based on the structure models presented here , we hypothesized that the N255K mutation uncouples nucleotide- and microtubule-binding functions in kinesin by compromising the ability of this residue to bridge between alpha tubulin and the backbone of E236 ( see Figure 4D , E ) , so eliminating communication between the microtubule and the switch loops . 10 . 7554/eLife . 04686 . 024Figure 7 . N255K uncoupling mutation compromises the microtubule interface , with accompanying orientational disorder of the motor domain . ( A ) , ( B ) Microtubule-stimulated ADP release kinetics measurements for the N255K mutant of our K349 construct . Shown in ( A ) are individual time traces , while ( B ) plots the fitted rate constants as a function of microtubule concentration . The extrapolated maximum rate for microtubule-stimulated ADP release was kmax = 0 . 26 ± 0 . 04 s−1 , with an apparent microtubule dissociation constant of K0 . 5 = 21 . 3 ± 6 . 3 μΜ . ( C–E ) Cryo-EM reconstructions of microtubules decorated with wild-type K349 ( Sindelar and Downing , 2010 ) and the N255K mutant . For the wild-type structure in ( C ) , the ADP state was selected for viewing purposes but all nucleotide states have similar features at this resolution . Maps from reconstructions of 14-protofilament microtubules are shown; features in the corresponding 13-protofilament reconstructions were very similar ( results not shown ) . All maps are low-pass filtered to 18 Å resolution , corresponding the estimated resolution of the N255K reconstructions . Supporting information describing cryo-EM analysis and 3D reconstruction of the N255K samples are shown in Figure 7—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 04686 . 02410 . 7554/eLife . 04686 . 025Figure 7—figure supplement 1 . Analysis of cryo-EM images of N255K kinesin complexed with microtubules ( see Figure 7 ) . ( A–D ) Compressed alignments ( similar to Figure 1—figure supplement 1D ) of representative single microtubules under various decoration conditions . A summation of the power spectra for every selected microtubule of the corresponding condition is shown to the right of each compressed microtubule image . All micrographs were collected under similar imaging conditions ( magnification , voltage , defocus , etc ) , using the same equipment ( FEI TF20 electron microscope equipped with a Gatan US4000 CCD camera ) . For comparison , analysis of an undecorated microtubule data set is shown in ( A ) . A green arrow indicates the location of the 80 Å layer line in these power spectra , present only in the cases where there is motor decoration . Compared with the wild-type K349 construct ( panel ( B ) ) , the N255K mutant ( panels ( C ) , ( D ) ) binds to microtubules but the 80 Å layer line due to motor binding is significantly weaker . The weaker 80 Å diffraction signal is indicative of poor ordering and/or occupancy of the attached motor heads . ( E–H ) , images formed by summing aligned box segments corresponding to the samples in ( A–D ) . Visible density for kinesin in these summations ( green arrows ) indicates that the N255K construct decorates microtubules with similar efficiency as the wild-type construct , despite weaker 80 Å diffraction by the mutant . This observation was supported by the ability of the N255K mutant to co-sediment with microtubules even in the ADP condition ( see ‘Materials and methods’ ) . Therefore , weak 80 Å diffraction in ( C ) and ( D ) must originate from heterogeneity in conformation of the bound motor heads rather than low occupancy , consistent with the reconstructions shown in Figure 4 of the main text . ( I ) Image of a microtubule decorated by the N255K construct ( ADP condition ) . While obviously decorated , the microtubule has a shaggy appearance uncharacteristic of decoration by wild-type kinesin . ( J ) Example of a morphologically disrupted microtubule , frequently seen in cryo-EM specimens of microtubules decorated by the N255K construct . The disrupted microtubules were excluded from data sets used for image processing and 3D reconstruction . DOI: http://dx . doi . org/10 . 7554/eLife . 04686 . 025 Cryo-EM analysis of the ADP and ATP analog-bound states of the N255K point mutant of our construct support this hypothesis , revealing two previously unidentified binding modes of kinesin in which the motor domain attaches tightly to the microtubule but is rotationally mobile ( Figure 7C–E and Figure 7—figure supplement 1 ) . The corresponding 3D maps did not refine to high resolution , presumably due to mobility of the kinesin heads . However , density for the motor domains in the N255K maps clearly indicates that a wild-type binding orientation is not achieved in either case . In the ADP-state map , the microtubule contact at site 255 appears to be attenuated or disordered ( Figure 7D , right panel ) , while in the AMPPNP map the wild-type microtubule contact of loop L12 is absent but a contact is evident at the 255 site ( Figure 7E , right panel ) . Thus , in neither nucleotide state is the N255K construct able to form all three wild-type microtubule contacts . These results indicate that the specific bridging role of N255 between the switch II loop and N413 of alpha tubulin ( see Figure 4 ) , which likely cannot be sustained by the N255K mutation , is not required for tight microtubule attachment . On the other hand , the N255K mutant appears to be incapable of maintaining a fixed orientation on the microtubule surface . We therefore propose that N255 acts as a conformational specificity selector , forcing the switch loops to close once kinesin contacts the microtubule . While the K255 substitution lacks the appropriate geometry to fulfill this selector role , the amino group of K255 would likely form non-specific interactions with the negatively charged microtubule surface . Presumably , flexibility in the resulting contact allows the N255K mutant to maintain affinity for microtubules even when the switch loops are open . However , failure of this mutant to close the switch loops under these conditions would lead to the loss of microtubule-stimulated ADP release and catalysis , consistent with experimental measurements .
It has increasingly been proposed that kinesin may harness the energy of twisting in its central beta sheet in some fashion during the ATPase cycle ( Hirose et al . , 2006; Kull and Endow , 2013; Arora et al . , 2014 ) . Speculations in this regard have mainly focused on the possibility , by analogy to myosin , that release of nucleotide initiates a much larger end-to-end twist in the sheet than is observed in comparisons of nucleotide-bound X-ray structures of the motor ( Gigant et al . , 2013; Kull and Endow , 2013; Arora et al . , 2014 ) . However , it has remained unclear how such twisting would relate to the complex interplay of affinities between kinesin and microtubules , ADP and ATP . The structure models derived in the current study indicate that in kinesin , the energy of nucleotide binding is captured by a related but distinct mode of distortion in the peptide backbone , involving coordinated rearrangements of the beta sheet and alpha 2 ( Figure 7 ) . As we have shown , this distortion is coupled with closure of the nucleotide cleft and can evidently be stabilized either by interactions between the P-loop and the open switch II loop ( ADP state; Figure 4F and Video 8 ) , or between the gamma phosphate and the closed switch loops ( ATP state; Figure 4E and Video 7 ) . This mechanism would thus allow microtubules to regulate kinesin's affinities for ADP and ATP by modulating the conformation of the switch loops , yielding an elegant structural basis for kinesin's nucleotide-binding behavior on and off microtubules . A number of sites in the kinesin fold have been identified that uncouple the motor's nucleotide affinity from its microtubule affinity , but the structural basis of the uncoupling has remained unclear . The nucleotide-exchange mechanism proposed here predicts that the uncoupling mutations in the kinesin's switch regions would have differential effects on the kinetics of ADP release , depending on the nature of the affected site . For example , due to its apparent role in stabilizing the nucleotide cleft in the detached motor ( see Figure 4F ) , perturbation of the E236 site would be expected to produce a comparatively large effect in kinesin's detached state , by destabilizing closure of the nucleotide cleft and increasing the ADP off rate . In contrast , mutation at the N255 site is not expected to produce a large effect in the detached motor but is expected to drastically attenuate microtubule-stimulated ADP release by compromising the ‘linchpin’ function of this residue . Both of these predictions are supported by kinetics measurements reported here and elsewhere , which reveal that the N255K mutation reduces the rate of microtubule-stimulated ADP release by up to 1000-fold , to near basal levels ( Auerbach and Johnson , 2005 ) ( Figure 7A , B ) , while the E236A mutation increases the ADP off-rate of detached kinesin by 100-fold ( Rice et al . , 1999 ) but does not significantly alter the rate of microtubule-stimulated ADP release . X-ray studies of the corresponding point mutants in KAR3 kinesin , co-complexed with ADP and magnesium , also support our interpretation . The structure of the KAR3 E631A mutant ( equivalent to E236A ) confirms that the mutated side chain can no longer interact strongly with the P-loop ( Yun et al . , 2001 ) , supplying an explanation for the increased ADP off rate in the detached state of this mutant . In contrast , the structure of KAR3 N650K ( equivalent to N255K ) was found to be essentially identical to that of wild-type ADP complex ( Yun et al . , 2001 ) , consistent with our proposal that the dominant functional effect originates in the microtubule complex rather than the detached state , for this latter mutant . The scheme in Figure 8 also provides a framework for understanding how gating can be achieved between the motor domains of dimeric kinesin ( Figure 8C ) . When both heads are attached to the microtubule in their tight-binding states , the neck linker of the trailing head is constrained in an arrangement that favors docking , thus tending to keep the docking cleft open ( Figure 8C , state 1 ) . In contrast , the neck linker in the leading head is kept out of its docking cleft , thus favoring closure of the cleft . Thus , in the trailing head , tight coupling between the docking cleft and the nucleotide cleft ( generated by the closed conformation of the switch loops ) keeps the nucleotide cleft closed . Meanwhile , the same coupling pathway keeps the nucleotide cleft open in the leading head—weakening the nucleotide affinity and thus disfavoring the binding and/or hydrolysis of ATP . Following hydrolysis and phosphate release in the trailing head ( Figure 8C , state 2 ) , however , strain generated at the active site in this head will no longer be supported by the nucleotide . At this point in the cycle , it appears that the neck linker would serve an important role as an allosteric modulator . If the neck linker were to be undocked following phosphate release , the trailing head would be free to revert to its previous conformation in the ATPase cycle ( Figure 8B , lower left ) and release ADP . However , if a forward step has occurred as depicted in states 1–2 , the neck linker in the trailing head will continue to be held in a docked conformation and the only recourse to relieve strain at the active site is to open the switch loops , hence opening the polymer cleft . The resulting ADP state of the trailing head thus favors tight nucleotide binding and weak microtubule affinity-promoting detachment from ( and discouraging subsequent reattachment to ) the trailing microtubule site ( Figure 8C , state 3 ) . Importantly , binding of ATP in the leading head at this point in the cycle ( Figure 8C , step 4 ) promotes a forward step by the trailing head through docking of the neck linker but does not prevent a backward step because neck linker docking is only weakly supported in the motor's ATP state ( Rice et al . , 2003 ) . Thus , the properties just described for the ADP-bound trailing head are likely critical in preventing back-stepping , which is a key determinant of efficient and rapid motility . Other structural schemes have been proposed in order to account for differences in nucleotide-binding and other properties between the leading and trailing heads of kinesin ( Hirokawa et al . , 2009; Sindelar , 2011 ) . In such schemes the conformation of the neck linker was mainly thought to affect the nucleotide affinity by triggering rearrangement of the switch loops . However , our structural data do not support this type of mechanism , instead indicating that the switch loops are held in a closed conformation by the microtubule in both leading and trailing heads providing the basis for the gating scheme in Figure 8C . The mechanism inferred here for kinesin reveals new parallels with the myosin molecular motor , which appears to be ancestrally related to kinesin ( Kull et al . , 1998 ) and shares very similar nucleotide-sensing ‘switch’ motifs ( Kull et al . , 1996; Sablin et al . , 1996 ) . While the structural basis of myosin's filament-attached power stroke is not well understood , it has been proposed that the corresponding lever-arm movement is coupled to progressive relaxation of the central beta sheet ( corresponding to increasing degrees of twist ) that would accompany catalysis and/or product release ( Coureux et al . , 2004 ) . This proposal mirrors the sequence of events we infer for kinesin , where the magnitude of the peptide backbone distortion appears to be greatest in the ATP state , less for the ADP states , and least of all for the no-nucleotide conformation ( Figure 6C and Figure 6—figure supplement 1C-F ) . A recent modeling study has also suggested that interactions with actin could lock myosin's switch I and switch II loops stably together while the P-loop moves with respect to them during the filament-attached power stroke ( Preller and Holmes , 2013 ) ; this behavior would be directly analogous to the behavior of microtubule-attached kinesin in the currently proposed scheme . An important functional difference between myosin and kinesin , however , is that these motors attach and detach from their partner filaments at different points in the ATPase cycle . In this light , it is worth noting that the ‘linchpin’ N255 residue in kinesin is conserved in myosin , but interacts natively with a conserved tyrosine found within the lower 50kD domain of myosin itself , rather than interacting with the filament as occurs with kinesin . Consequently , in myosin this asparagine holds the switch II loop near its ‘closed’ conformation even in the absence of actin , which could help explain why myosin , unlike kinesin , shows strong catalytic activity even in the absence of its partner filament ( Geeves and Holmes , 2005 ) . We anticipate that future studies of both kinesin and myosin will shed more light on the relationship between these intriguing molecular machines . Concurrently with the current work , an X-ray crystal structure of no-nucleotide kinesin in complex with a non-polymerized tubulin dimer has been published ( Cao et al . , 2014 ) . The structure of no-nucleotide kinesin reported in the X-ray study is in excellent agreement with model presented here ( backbone RMSD for all visible residues is 1 . 28Å ) . Accordingly , the detailed interactions of kinesin and tubulin seen in the X-ray structure appear to be fully consistent with the models and mechanism presented in the current work .
The wild-type , monomeric human K349 construct was bacterially expressed and purified as described ( Kull et al . , 1996 ) , and 15% ( wt/vol ) sucrose was added before snap freezing in liquid nitrogen and storing at −80°C . A plasmid for the mutant N255K construct was generated from the wild-type plasmid using the QuikChange site-directed mutagenesis kit ( Agilent Technologies; Santa Clara , CA ) . Thawed K349 ( either wild-type or N255K ) was exchanged into EM buffer ( 25 mM PIPES , 25 mM KCl , 1 mM EGTA , 1 mM DTT ) using three rounds of dilution and concentration in a Microcon ultracentrifugal filter ( EMD Millipore; Billerica , MA ) . Microtubule batches were grown from 250 µg of lyophilized bovine brain tubulin ( Cytoskeleton; Denver , CO ) , resuspended in 25 µl EM buffer and clarified ( Beckman TLA 120 . 2 , 100K RPM , 4°C ) prior to incubation at 37°C . Taxol ( 2 mM in DMSO ) was added to equimolar levels with tubulin after 10 min of incubation . Following ∼45 min of polymerization , microtubules were brought to room temperature and a ∼twofold excess of K349 was added prior to pelleting through a glycerol cushion ( 50 µl of EM buffer + 60% glycerol wt/vol + 200 µM taxol ) in order to remove unbound motor and unpolymerized tubulin ( 20 min , Beckman TLA 120 . 2 , 50K RPM , 24°C ) . The motor–microtubule complex was resuspended in ∼10 µl of EM buffer plus 200 µM taxol . Stopped-flow measurements were performed at 298 K using an SF-300X ( Indiana University ) stopped-flow apparatus ( KinTek Corp . ; Austin , TX ) equipped with a Xenon arc lamp ( Hamamatsu; Japan ) . Kinetics of the interaction of mant-ADP with kinesin was measured by equilibrating a kinesin·mant-ADP ( 1:1 ) complex followed by rapid mixing with a high concentration of MgATP or varying MT concentrations plus MgATP to chase the mant-ADP from the active site as described ( Sadhu and Taylor , 1992 ) . Mant fluorescence was monitored over time , Iex , max = 356 nm , Iem , max = 448 nm ( 400 nm long-pass filter ) . Samples were plunge-frozen in liquid ethane , using Quantifoil holey carbon grids with 1 µm hole diameter , 1 . 5 µm spacing ( Quantifoil Micro Tools GmbH; Germany ) . In order to optimize motor decoration on the microtubules , glow discharge was not applied to the grids and samples were diluted ∼5–10× into distilled water ( 0 . 35 µl sample plus 3 µl d ( H2O ) ) on a piece of Parafilm prior to grid application , in order to compensate for evaporation that occurs prior to contact with liquid ethane ( Sindelar and Downing , 2007 ) . For the ADP , AMPPNP , and ADP•Al•Fx conditions , the grid droplet mixture was supplemented by 2 mM nucleotide ( 2 mM ATP + 2 mM AlCl3 + 10 mM NaF for the latter condition ) . Following our previously published protocol ( Sindelar and Downing , 2007 ) , most of the initially applied buffer on the grids was wicked away by touching the grid edgewise with a piece of filter paper ( Whatman ) . The grids were then blotted completely , and after a 0 . 5–1 s delay , immersed into liquid ethane using a home-built plunge-freezing apparatus . Consistent with our prior experience ( Sindelar and Downing , 2007 ) , failure to dilute the sample buffer ∼5–10× prior to plunge freezing led to poor and/or inconsistent kinesin decoration of the microtubules . Micrographs of the microtubules decorated by the wild-type K349 construct were collected using the SerialEM package to collect data semi-automatically on 300 kV FEG-equipped electron microscopes ( FEI F30 for the no-nucleotide data set , FEI Titan for the ADP•Al•Fx data set ) , using K2 direct electron detecting cameras ( Gatan; Pleasanton , CA ) in video mode . For bare microtubules , and microtubules decorated by the N255K construct , micrographs were collected using a TF-20 FEG-equipped instrument and a Gatan US4000 CCD detector . In all cases , the defocus was systematically varied from approximately 1 to 2 . 5 µM through the course of the data collection . The total dose was ∼15 electrons/Angstrom squared , distributed over 15 or 16 video frames . The no-nucleotide data set was collected at ∼13K magnification using the camera's super-resolution mode and subsequently binned 2× , yielding 4K by 4K image dimensions with an effective pixel size of 1 . 99 Å . The ADP•Al•Fx data set was collected at approximately the same magnification as the no-nucleotide data set but in regular counting mode , resulting in 4K by 4K images with an effective pixel size of 2 . 097 Å . After performing drift analysis ( Li et al . , 2013 ) , whole-image video frames were aligned and averaged ( K2 data only ) before manually selecting overlapping boxed segments corresponding to individual microtubules . Defocuses and astigmatism parameters were estimated using the CTFFIND3 program ( Mindell and Grigorieff , 2003 ) . Single-particle analysis and 3D reconstruction was then performed as described ( Sindelar and Downing , 2010 ) , with some modifications . Initial reference models were generated by applying a parametric model of the microtubule ( Chrétien and Wade , 1991 ) to PDB models of the no-nucleotide kinesin-microtubule complex ( Sindelar and Downing , 2007 ) , converting the atomic coordinates to EM density using the SPIDER package ( Frank et al . , 1996 ) , and applying a low-pass filter with a 20 Å cutoff frequency . Multi-reference analysis performed using a range of different microtubule assemblies ( 12–15 protofilaments ) established that 14-protofilament microtubules outnumbered 13-protofilament by approximately 3:1 , with only marginal populations of other symmetry forms . Based on this determination , the 13- and 14-protofilament sub-populations were separately selected for further analysis . Initial estimates of the in-plane rotation of individual segments were made using the Radon transform , as described ( Li et al . , 2002 ) . Automated scripts using the SPIDER package ( Frank et al . , 1996 ) were used for semi-exhaustive searching of XY shifts and Euler angles ( out-of-plane tilt range: +/−15° ) . Reference alignment was used to determine the position of the microtubule seam on a per-microtubule basis as described ( Sindelar and Downing , 2007 ) , as was the filament polarity , and then every segment was subjected to local refinement using the established seam orientation and polarity . Evidently due to disorder in the N255K kinesin head orientations , the correlation scores used to identify the seam orientation were far noisier for this mutant . Following initial refinement of the Euler angles and shifts for each box , the estimated position of each 8 nm repeat of the microtubule was mapped back onto the micrograph and a new stack of boxes was extracted for a final round of SPIDER refinement . Thus , the final data set included exactly one box for each 8 nm repeat identified in the selected microtubules . Subsequent structure refinement and 3D reconstruction was performed using the FREALIGN package ( Grigorieff , 2007 ) with specific modifications for helical image processing ( Alushin et al . , 2010 ) . For the FREALIGN refinements , four rounds of refinement and reconstruction were initially performed , using successively higher resolution cutoffs for the refinement target ( 20 Å , 15 Å , 12 Å , 10 Å ) . For each reconstruction , 13- or 14-fold pseudo-helical symmetry was applied in order to transform all imaged asymmetric subunits onto a single ‘good’ protofilament , as previously described ( Sindelar and Downing , 2007 ) . Helical parameters were derived from the corresponding , canonical microtubule form ( Chrétien and Wade , 1991 ) but adapted for the measured axial repeat distance in our data sets . The reconstructed ‘good’ protofilament ( along with the remainder of the reconstruction ) was subsequently replicated and transformed 13 or 14 times using the same symmetry parameters in order to generate protofilament models for the entire microtubule . The transformed ‘good’ protofilaments were subsequently selected by complementary wedge-shaped masks and summed in order to obtain the final asymmetric microtubule model . To reduce the influence of solvent noise in the refinement , a tight mask around the protein density was generated from a thresholded low-resolution version of the current map , and then smoothed with a soft Gaussian filter ( ∼10 Å half-width ) . This mask was then applied to the reconstructed volume prior to the next round of refinement . The same mask was employed for FSC calculations . Once the FREALIGN steps were completed , the resulting 3D map was then filtered to 20 Å and fed back into the SPIDER/FREALIGN pipeline , in order to reduce errors related to seam identification , polarity , and local searches . This additional step was omitted from the refinement of the N255K mutants . Following the second cycle of SPIDER/FREALIGN analysis , FSC calculations indicated that the resolution of the wild-type reconstructions was approximately 6 Å ( 0 . 143 criterion; Figure 1—figure supplement 1A , B ) , and no signs of higher-resolution features were apparent . We then subdivided the aligned video frames corresponding to each micrograph into summed groups of three ( 5 frames each ) , and re-extracted a new FREALIGN image stack from these sub-frames , resulting in a threefold larger number of stacked images . We then performed four more rounds of FREALIGN analysis with the new stack , using Euler angles and shifts determined previously as starting parameters . The resulting reconstructions resolved individual beta strands and alpha helical pitch in some portions of the map ( Figure 1—figure supplement 1F , G ) , although as noted in the main text the kinesin density was more poorly resolved and there was evidence of anisotropic blurring in the tubulin density ( results not shown ) . The final resolution , obtained through FSC comparison of reconstructed half-data set volumes after applying a soft mask , was ∼5 Å ( 0 . 143 criterion ) for both nucleotide states ( Figure 1—figure supplement 1A , B ) . For the final no-nucleotide reconstruction , a total of 10 , 029 8 nm repeats were used , for a total of ∼140000 asymmetric units ( 14 protofilaments ) . A total of 2394 8 nm repeats , for a total of ∼33 , 600 asymmetric units , were used for the final ADP•Al•Fx reconstruction ( 14 protofilaments ) . In order to derive an atomic model for the microtubule complex of ATP-bound kinesin , we subjected the coordinates of tubulin and kinesin from the 4HNA structure to a hybrid all-atom molecular dynamics method ( MDFF ) in which cryo-EM density contributes a force field term ( steering potential ) designed to guide the simulation toward the experimentally observed structure ( Trabuco et al . , 2008 ) . Version 1 . 91 of NAMD was used for all molecular dynamics calculations ( Phillips et al . , 2005 ) . We used an explicit solvation model ( TIP3P ) , because conformational instabilities were observed in trial simulations that used a less expensive continuum solvent model . All simulations were run using periodic boundary conditions , but no attempt was made to model the longitudinal or lateral interfaces of tubulin . While this omission likely produced artifacts near the boundaries between the simulated tubulin heterodimer and neighboring tubulin subunits , these boundaries are remote from the motor–microtubule interface . Moreover , the EM steering potential used in these simulations conformation strongly clamps the conformation of tubulin . Thus , our simulation setup is expected to minimize or eliminate the propagation of any boundary artifacts toward kinesin and its interface with tubulin , which is the focus of the current study . To complete the starting model , twenty water molecules were manually placed within the kinesin nucleotide pocket at sites corresponding to crystallographic waters identified in a high-resolution ( 1 . 7 Å ) X-ray structure of KIF4 kinesin co-complexed with AMPPNP ( Chang et al . , 2013 ) . Hydrogen atoms were added to protein atoms and crystallographic water molecules using the ‘guesscoord’ command from the NAMD package ( Phillips et al . , 2005 ) . The system was then placed in a TIP3P water box of dimensions 125 × 125 × 95 Å , and sodium and chloride ions were randomly added to this water box to a concentration of 50 μM , at a ratio that neutralized the charge of the system . After energy minimization ( 600 steps ) , the system was subjected to a three-phase equilibration , in which protein atoms were initially subjected to positional restraints that were then progressively released . The equilibration simulations were performed in the NPT ensemble , using a Nosé–Hoover Langevin piston with a target pressure of 1 atm , a decay period of 200 fs and a time constant of 100 fs . The temperature was maintained at 310 K using a Langevin temperature bath with a time constant of 5 ps−1 . In the first equilibration phase , which ran for 50 ps , all protein and ligand atoms were restrained by a 20 kcal/mol/Å harmonic potential; the second equilibration step restrained protein backbone atoms only and the nucleotide ribose rings ( 50 ps ) ; and the final step removed all harmonic constraints and was run for 600 ps . For all simulations , the CHARMM27 force field was used , with long-distance electrostatic interactions computed by the particle-mesh Ewald method and a non-bonded cutoff distance of 10 Å . Force field terms for GDP and GTP were adapted from ADP and ATP , respectively . Following equilibration , we performed MDFF simulations , but with substantial modifications in order to reduce the possibility of overfitting , which has been shown to be a significant concern with this method ( Bai et al . , 2013 ) . Importantly , we limited the influence of the EM steering potential to include only protein backbone atoms and nucleotide ligands . Thus , side chain atoms in our MDFF simulations are subjected exclusively to physics-based force field terms , which is reasonable because side chains were not visualized in our maps . Related to this point , as noted in the main text , a low-pass filter was used to remove signal beyond 6 Å resolution in the cryo-EM map , in order to reduce the level of contaminating noise prior to generating the EM steering potential . Limiting the EM steering potential to backbone atoms tends to moderate the contribution of the EM term in the simulated energy function , thus compensating for the limited number of experimental constraints available from our cryo-EM maps . In conventional applications of MDFF , the EM steering potential is introduced as a step function after an initial equilibration period , and the relative strength of the potential ( compared with the physics-based terms in the molecular dynamics force field ) is described by an adjustable parameter , ξ , whose magnitude must be empirically determined . If the value of ξ is too low , it will not force a conformational transition within an achievable amount of simulation time; on the other hand , if the value is too high the molecular conformation becomes distorted ( ‘overfitting’ ) . However , a quantitative method for choosing an appropriate value for the ξ parameter has not been presented . To address this problem , we developed a modified MDFF protocol in which the value of ξ follows a linear ramp function , starting at zero and slowly increasing over the course of 10 nanoseconds . We then monitored the system for convergence by calculating the root mean squared deviation between the coordinates of the fitted structure and the starting model ( Figure 2—figure supplement 1 ) . The resulting simulation trajectory exhibits a rapid conformational transition that occurs within the first 2 nanoseconds of the simulation , corresponding to relatively low values of the GSCALE parameter ( GSCALE <0 . 15 , compared with the ‘typical’ recommended value of 0 . 3 [Trabuco et al . , 2008] ) . The RMSD signal was dominated by a large conformational change in tubulin , increasing to over 2 . 5 Å by the 2 ns time point ( Figure 2—figure supplement 1B ) . This initial transition is followed by a gradual increase in the reported backbone RMSD values in both kinesin and tubulin that continues to the end of the simulation ( GSCALE = 1 . 0 ) . Inspection of the simulation trajectory reveals that the initial transition mainly consists of a straightening of the tubulin subunits from the conformation seen in 4HNA in order to accommodate the microtubule lattice . This transition is essentially complete after 1 ns of simulation time ( see Video 1 ) . In contrast to tubulin , the conformation of kinesin is largely unaffected during the first 2 ns of the MDFF simulation , and the backbone RMSD values ( kinesin only ) remain at approximately 1 . 0 Å with respect to the starting conformation for this period . Subsequent to the first 2 ns of the MDFF simulation , the protein backbone progressively becomes disrupted beyond a level that can be justified by observable features in our EM density maps . A prominent example is loop L9 from kinesin , which encloses the nucleotide pocket and includes the switch I motif . In the starting model , a network of hydrogen bonds supports the structure of this loop; however , by the end of the simulation many of these interactions are lost as the loop is increasingly ‘squeezed’ into the corresponding density feature ( results not shown ) . We note that the path of the protein backbone is not directly resolved this region of our density map , which means that the corresponding EM potential tends to uniformly pull backbone atoms toward the center of the loop feature ( where density values are highest ) , and thus away from their true path . Disruption of L9 ( and other secondary structure elements ) in the latter parts of the ATP-state MDFF simulation is therefore likely due to over-fitting as the EM steering term increasingly predominates over the other energy terms in the simulated potential . We also note that the conformation of kinesin's N-terminal segment ( residues 1–8 ) , termed the neck cover strand ( Khalil et al . , 2008 ) , is unstable even in early time points of the MDFF simulation , possibly due to weak density for both this element as well as the neck linker in the cryo-EM map . Limited occupancy of the docked neck linker is in fact expected under the experimental conditions used here ( Rice et al . , 2003 ) . In order to minimize the presence of over-fitting artifacts in our final structure model , we selected an early time point ( t = 1 . 2 ns ) that immediately follows the initial transition , in order to represent the ATP-bound kinesin-microtubule complex ( Figure 2—figure supplement 1B ) . The stability of the resulting structure model was investigated using a follow-up simulation in which the EM steering potential was turned off but the conformation of tubulin was subjected to harmonic positional restraints ( 20 kcal/mol/Å ) in order to maintain the straight conformation of tubulin found in the microtubule lattice . Only residues 1–380 of alpha and beta tubulin were included in the restraint potential , thus excluding the kinesin-binding regions ( helices H11-H12 ) . As shown in Figure 2—figure supplement 1D , the conformation of kinesin was relatively stable over the course of the 2 ns time period investigated , maintaining a backbone RMSD of ∼1 Å from the starting structure . Signature hydrogen-bond interactions of the closed switch loop network ( Y138 , R203 , E236 , E250 , N255 ) were maintained throughout this simulation , with the notable exception of the conserved salt bridge between R203 and E236 , conserved in all X-ray structures where the switch loops are closed , which dissociated partway through the simulation ( results not shown ) . This effect was also seen in unconstrained simulations of the 4HNA crystallographic model ( results not shown ) , indicating a possible deficiency in the CHARMM27 energy function and/or initial structure model used here . In order to obtain an atomic model for kinesin's no-nucleotide state on microtubules , we repeated the above simulation protocol but replaced the target function with one derived from the no-nucleotide cryo-EM map . After modifying the 4HNA coordinates as described in the main text ( deletion of neck-linker residues and ATP ) , we equilibrated this system to allow water into the newly vacated nucleotide pocket , and then introduced an cryo-EM steering potential ( linear ramp over 10 ns , as was done for the ATP state model ) in order to bias the coordinates toward the conformation observed in our no-nucleotide map . Similar to the ATP state trajectory , we observed a rapid conformational transition in the early phase of the MDFF simulation that was essentially complete by 1 ns ( GSCALE = 0 . 2 ) ( see Video 2 ) . The behavior of tubulin in this trajectory , as reflected by the backbone RMSD from the starting structure , was also very similar to that seen in the ATP trajectory . The backbone RMSD values for kinesin , however , were much greater in the no-nucleotide trajectory , reflecting a significant conformational rearrangement . As described in the main text ( Figure 3 ) , the primary structural changes in kinesin were twisting of the N-terminal , upper , and lower subdomains with respect to each other , followed by separation of the P-loop from the switch II loop and attendant reorientation of the K91 side chain . The subdomain twisting movement was completed somewhat before the P-loop fully separated from the switch II loop , resulting in elongation of the P-loop as well as distortion of the closed switch loop network prior to the separation . After these elements had separated , however , they both ‘snapped’ back to conformations that resembled those seen in the starting model ( Video 3 ) . Similar to the behavior of tubulin in our simulations of both nucleotide states , the kinesin transition was largely complete by 1 ns , as reflected by the backbone RMSD from the starting structure ( Figure 2—figure supplement 1A ) as well as the observed interactions in the atomic model itself ( Video 3 ) . As with the ATP-state model , we therefore sought to avoid overfitting artifacts by selecting a trajectory frame immediately following the rapid transition ( t = 1 . 4 ns ) to represent the conformation of no-nucleotide , microtubule-complexed kinesin found in our maps . Generally , the agreement between the resulting atomic model and the cryo-EM map is excellent . The only obvious discrepancy between the map and the model is found in the vicinity of residues ∼195–200 at the tip of L9 , which forms part of the switch I response element . In this region , a substantial reduction in the average density of the no-nucleotide cryo-EM map indicates the presence of disorder . Consistent with this interpretation , we observed a corresponding increase in conformational fluctuations at the tip of L9 in a follow-up molecular dynamics simulation where the cryo-EM potential was turned off ( See below ) , while the remainder of the active site remained firmly locked in place ( Figure 2—figure supplement 3A ) . We used the selected model from our no-nucleotide MDFF simulation to perform a 2 ns follow-up simulation where the EM steering potential was eliminated and the tubulin coordinates harmonically constrained , mirroring the protocol used for the ATP-state ( above ) . This trajectory maintained the entire network of closed switch loop hydrogen bonds ( Y138 , R203 , E236 , E250 , N255 ) found in the starting structure , including the salt bridge between R203 and E236; the active-site salt bridge formed between K91 and D231 in the starting model were also maintained throughout the trajectory ( results not shown ) . These observations indicate that the no-nucleotide structure identified by our flexible fitting experiments is at least transiently stable . However , in this follow-up simulation kinesin exhibited substantially more flexibility ( ∼1 . 5 Å RMSD ) compared with the corresponding ATP-state trajectory ( compare Figure 2—figure supplement 1C , D ) . This effect could be a consequence of the open conformation of the nucleotide cleft , and loss of attendant interactions between the switch II and the P-loop , seen in the no-nucleotide state structure . All figures and videos were rendered with UCSF Chimera ( Pettersen et al . , 2004 ) . Cryo-EM maps and coordinates for no-nucleotide and ADP•Al•Fx states of the kinesin-microtubule have been deposited in the EMDB ( accession codes 6187 and 6188 , respectively ) and PDB ( ID codes 3J8X and 3J8Y , respectively ) .
Microtubule-based transport by the kinesin motors , powered by ATP hydrolysis , is essential for a wide range of vital processes in eukaryotes . We obtained insight into this process by developing atomic models for no-nucleotide and ATP states of the monomeric kinesin motor domain on microtubules from cryo-EM reconstructions at 5–6 Å resolution . By comparing these models with existing X-ray structures of ADP-bound kinesin , we infer a mechanistic scheme in which microtubule attachment , mediated by a universally conserved ‘linchpin’ residue in kinesin ( N255 ) , triggers a clamshell opening of the nucleotide cleft and accompanying release of ADP . Binding of ATP re-closes the cleft in a manner that tightly couples to translocation of cargo , via kinesin's ‘neck linker’ element . These structural transitions are reminiscent of the analogous nucleotide-exchange steps in the myosin and F1-ATPase motors and inform how the two heads of a kinesin dimer ‘gate’ each other to promote coordinated stepping along microtubules .
The inside of a cell is a dynamic environment . Large molecules such as proteins are commonly transported within a cell by ‘motor proteins’ , which move along a network of filaments called microtubules . One group of motor proteins , the kinesins , typically have one end called a motor domain that attaches itself to a microtubule . The other end links to the cargo being carried , and a flexible ‘neck’ region connects the two ends of the motor protein . Kinesins are bound together in pairs . The flexible neck region allows each motor domain in a pair to move past that of the other , allowing the kinesin to ‘walk’ along a microtubule in a step-like manner . Each step requires one motor domain to alternately tightly associate with , and then release from , a microtubule filament . This alternating cycle is coordinated by kinesin binding to and breaking down a molecule called ATP to form another molecule called ADP , which releases the energy needed for its next step . This repeating cycle is possible because a motor domain changes shape when it binds to a microtubule . This shape change stimulates the release of ADP , freeing up room for a new ATP molecule to bind to the motor domain . Although relatively small , these structural changes produce larger changes in the flexible neck region that enable the individual motor domains within a kinesin pair to co-ordinate their movement and move efficiently . Many previous studies have investigated these shape changes using a technique called cryo-electron microscopy , which rapidly freezes samples and allows their structure to be recorded in high detail . However , the small size of the motor domains and their changes in shape means that this technique was not able to reveal the structures in full detail . Shang et al . now exploit recent advances in cryo-electron microscopy to examine the structural changes of individual kinesin motor domains in greater detail . Images of motor domains bound to microtubules were made while the motor domain was in one of two different states: not bound to ATP or ADP , or bound to a chemically modified form of ATP that cannot be broken down . Shang et al . then used these images to produce models of the motor domains and compared the models with previously published images . This revealed a cleft in the kinesin motor domain that opens when it attaches to a microtubule . This cleft's ‘clamshell-like’ opening allows ADP to be released; it then closes when a molecule of ATP binds to it . The opening and closing of the cleft causes the changes in the ‘neck linker’ of the kinesin that enable the motor protein to transport its cargo , and so links ATP binding to the movement of the motor protein . Shang et al . suggest that similar processes may also occur in other motor proteins that have not been as well studied as the kinesins .