Kinesin Motor Mechanics: Binding, Stepping, Tracking, Gating, and Limping
2007; Elsevier BV; Volume: 92; Issue: 9 Linguagem: Inglês
10.1529/biophysj.106.100677
ISSN1542-0086
Autores Tópico(s)Cellular Mechanics and Interactions
ResumoThis critical review was motivated by the 10th Biophysical Discussions meeting, “Molecular Motors: Point Counterpoint”, held in Asilomar, CA, during October 19–22, 2006. Biophysical Discussions are meetings that focus on cutting-edge or emerging topics in biophysics that can benefit from intense discussions. Streaming videos of the speaker presentations at this conference, including a synopsis of this review, are available through the Biophysical Society’s web site at http://www.biophysics.org/discussions. In keeping with the spirit of a discussions meeting, I present here a personal perspective on the current state of kinesin motor mechanics. Nearly a generation has passed since the discovery of the motor named kinesin (1.Vale R.D. Reese T.S. Sheetz M.P. Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility.Cell. 1985; 42: 39-50Abstract Full Text PDF PubMed Scopus (1412) Google Scholar), and the subsequent development of the very first single-molecule gliding-filament and bead assays for motility (2.Howard J. Mechanics of Motor Proteins and the Cytoskeleton. Sinauer Associates, Sunderland, MA2001Google Scholar, 3.Block S.M. Goldstein L.S. Schnapp B.J. Bead movement by single kinesin molecules studied with optical tweezers.Nature. 1990; 348: 348-352Crossref PubMed Scopus (851) Google Scholar), which helped to establish the modern field of single-molecule biophysics. Discrete steps of single molecules were first measured for kinesin (4.Svoboda K. Schmidt C.F. Schnapp B.J. Block S.M. Direct observation of kinesin stepping by optical trapping interferometry.Nature. 1993; 365: 721-727Crossref PubMed Scopus (1586) Google Scholar), followed shortly thereafter by reports of similar steps for myosin (5.Finer J.T. Simmons R.M. Spudich J.A. Single myosin molecule mechanics: piconewton forces and nanometre steps.Nature. 1994; 368: 113-119Crossref PubMed Scopus (1569) Google Scholar, 6.Ishijima A. Harada Y. Kojima H. Funatsu T. Higuchi H. Yanagida T. Single-molecule analysis of the actomyosin motor using nano-manipulation.Biochem. Biophys. Res. Commun. 1994; 199: 1057-1063Crossref PubMed Scopus (175) Google Scholar). Since then, literally thousands of single-molecule experiments have been performed on a whole variety of molecular motors, all with the aim of discovering how these remarkable protein machines function. Considerable and impressive progress has been achieved, but key questions still abound, and this remains a very lively field of endeavor. I discuss below my current thinking on several questions concerned with kinesin mechanics, listed in no particular order of precedence. I wade into controversy holding no illusions that everyone will share my views on the answers to these questions, but I do hope to provoke a more thoughtful examination, and set the record straight on at least a few points. By choice, and in keeping with the topic of the meeting session where this was presented (“Motor Walking Mechanisms”), the questions that I’ve posed relate directly to the nanoscale mechanics of kinesin motion. However, these same questions are intimately and inevitably linked to other aspects of kinesin structure, biochemistry, and cellular function. In our original article describing single kinesin stepping, the steps were found to subtend a distance of 8 nm, and they took place instantaneously on the time scale of the experiment. Here, the data acquisition rate was 1 kHz (after anti-alias filtering at the Nyquist frequency, 0.5 kHz), and records were software-filtered to 200 Hz, for a characteristic time of 5 ms (4.Svoboda K. Schmidt C.F. Schnapp B.J. Block S.M. Direct observation of kinesin stepping by optical trapping interferometry.Nature. 1993; 365: 721-727Crossref PubMed Scopus (1586) Google Scholar). Quite a number of models to explain kinesin motion have since been entertained, which predict that the 8-nm step should be composed of substeps of one form or another. Substeps are by no means unreasonable to contemplate, for a variety of plausible reasons (see below). Two studies have claimed to identify substeps within the kinesin cycle. I don’t believe that either article presented a sufficiently compelling case that substeps exist. In both instances, there appear to have been similar flaws in methodology. The first article, a collaborative effort by Vale and Spudich (7.Coppin C.M. Finer J.T. Spudich J.A. Vale R.D. Detection of sub-8-nm movements of kinesin by high-resolution optical-trap microscopy.Proc. Natl. Acad. Sci. USA. 1996; 93: 1913-1917Crossref PubMed Scopus (104) Google Scholar) reported the existence of a comparatively long-lived intermediate state during the forward step, lasting on the order of 10–20 ms, which separated the 8-nm step into two distinct components of 5- and 3-nm (with the 5-nm component being the most clearly resolved). However, the starting and ending points of the steps in data records were a), scored entirely “by eye” from b), traces filtered with a 15-ms median filter. Under these circumstances, no statistically meaningful plateaus can exist whose characteristic times are comparable to that of the smoothing filter (15 ms). Although the data were sampled at 2 kHz, this did not mean that they were trustworthy at a data interval of 0.5 ms, because the bandwidth of the analog position signal was limited to 110 Hz, corresponding to a characteristic time of 9 ms. In retrospect, it seems likely that the milliseconds-long plateaus seen in the noisy records were the consequence of a data selection artifact. Since 1996, the time resolution for the routine recording of kinesin stepping has steadily improved, particularly for smaller beads subjected to higher loads, where it now routinely achieves ∼1 ms or better (see, for example, (Guydosh and Block (8.Guydosh N.R. Block S.M. Backsteps induced by nucleotide analogs suggest the front head of kinesin is gated by strain.Proc. Natl. Acad. Sci. USA. 2006; 103: 8054-8059Crossref PubMed Scopus (109) Google Scholar)). No group has ever duplicated these findings. The second article, the result of a collaborative effort by the Yanagida and Higuchi labs (9.Nishiyama M. Muto E. Inoue Y. Yanagida T. Higuchi H. Substeps within the 8-nm step of the ATPase cycle of single kinesin molecules.Nat. Cell Biol. 2001; 3: 425-428Crossref PubMed Scopus (133) Google Scholar), achieved substantially higher temporal resolution, and reported substeps lasting on the order of 50 μs, some 200-fold faster than those reported by Coppin et al. (7.Coppin C.M. Finer J.T. Spudich J.A. Vale R.D. Detection of sub-8-nm movements of kinesin by high-resolution optical-trap microscopy.Proc. Natl. Acad. Sci. USA. 1996; 93: 1913-1917Crossref PubMed Scopus (104) Google Scholar). Here again, though, the same two issues resurface, associated with a), data sampling by selection and b), a failure to assess the effects of instrument bandwidth. Because of the presence of noise, individual records of steps showed no clear evidence of substeps. However, a subset of records displayed small fluctuations (seen as plateaus) during their rising phase for a step: these records were separated from those that rose more smoothly (again, “by eye”) and placed in two further batches, with plateaus lasting either 50–100 μs or >100 μs, then separately averaged together. Such a selection procedure, followed by averaging, seems guaranteed to reinforce any random fluctuations (noise) that may have contributed to the plateaus, along with genuine signals (if any). The traces with apparent 50–100 μs plateaus seemed to divide the 8-nm step into two equal components of 4 nm. However, although data were acquired at 100 kHz using dark-field laser illumination onto a quadrant photodetector, signals had been passed through a 20 kHz analog low-pass filter before digitizing, so the characteristic response time of the measurements was 50 μs. This time is remarkably similar to their measurement of the average time constant for the abrupt rising phase of a step (Fig. 3 of Nishiyama et al. (9.Nishiyama M. Muto E. Inoue Y. Yanagida T. Higuchi H. Substeps within the 8-nm step of the ATPase cycle of single kinesin molecules.Nat. Cell Biol. 2001; 3: 425-428Crossref PubMed Scopus (133) Google Scholar)), which came to 48 μs. It is not meaningful to extract timing information in the “10 μs range” when instrument response times are restricted to comparable intervals. More recently, Cross’s group has reinvestigated the question of kinesin substeps, and reported finding no evidence for these down to their experimental cutoff time, estimated to at ∼30 μs (10.Carter N.J. Cross R.A. Mechanics of the kinesin step.Nature. 2005; 435: 308-312Crossref PubMed Scopus (568) Google Scholar). In their case, the measurement system, based on bright-field imaging onto a quadrant photodetector, had a combined bandwidth of 46 kHz (∼21 μs), but in most cases data were sampled at 80 kHz and averaged down to 20 kHz (∼50 μs) for analysis. The effective bandwidth is therefore quite comparable to that of the instrument employed by the Yanagida and Higuchi group. However, steps were scored here by an automated algorithm, and not binned by eye into categories for subsequent averaging. My own group has also sought evidence of kinesin substeps. In unpublished work, we found no evidence for these with an instrument that uses back focal plane detection of scattered laser light onto a position-sensitive detector. Our photodetection subsystem has an analog bandwidth of ∼200 kHz, but the computer data acquisition was limited to ∼35 kHz, corresponding to a characteristic system response time of ∼30 μs. We concur with Carter and Cross (10.Carter N.J. Cross R.A. Mechanics of the kinesin step.Nature. 2005; 435: 308-312Crossref PubMed Scopus (568) Google Scholar) that no substeps can be found down to this response time, and steps are still instantaneous on the timescale of our measurements. None of this is to say, however, that kinesin substeps don’t exist! The Yanagida group has argued that the size of the “characteristic distance”, δ, for kinesin movement (11.Schnitzer M.J. Visscher K. Block S.M. Force production by single kinesin motors.Nat. Cell Biol. 2000; 2: 718-723Crossref PubMed Scopus (467) Google Scholar), a parameter that can be derived from force velocity curves, implies the existence of substeps, given that its value is ∼3 nm, which is only a fraction the full 8-nm step (12.Nishiyama M. Higuchi H. Yanagida T. Chemomechanical coupling of the forward and backward steps of single kinesin molecules.Nat. Cell Biol. 2002; 4: 790-797Crossref PubMed Scopus (253) Google Scholar). However, I do not accept that argument as being decisive. As we have previously noted in Wang et al. (13.Wang M.D. Schnitzer M.J. Yin H. Landick R. Gelles J. Block S.M. Force and velocity measured for single molecules of RNA polymerase.Science. 1998; 282: 902-907Crossref PubMed Scopus (747) Google Scholar), the physical interpretation of the characteristic distance, δ, is highly model-dependent, and several very different classes of biochemical pathways can lead to force-velocity relationships with a similar Boltzmann-type shape. In some of these pathways, the characteristic distance corresponds directly to a measurement of the step size (14.Abbondanzieri E.A. Greenleaf W.J. Shaevitz J.W. Landick R. Block S.M. Direct observation of base-pair stepping by RNA polymerase.Nature. 2005; 438: 460-465Crossref PubMed Scopus (675) Google Scholar), but in others, it corresponds instead to the distance to a transition state, which is always less than the step size. It therefore seems possible that a value of δ ∼3 nm could be reconciled with either full-stepping or substepping pathways; additional evidence is required to decide the issue. Could substeps be accommodated? Yes, provided these are exceedingly short-lived. An unloaded kinesin head can diffuse over 8 nm in a time of ∼10 μs (based on approximating the head as a 10-nm diameter sphere diffusing in water through 8 nm, according to 〈x2〉 ≈ 2Dt). However, this first-passage time rises exponentially fast when the head is forced to move against a load of any size (15.Howard J. Hudspeth A.J. Vale R.D. Movement of microtubules by single kinesin molecules.Nature. 1989; 342: 154-158Crossref PubMed Scopus (742) Google Scholar). If the actual kinesin step consists, for example, of a), an initial conformational change followed by b), a diffusional component that carries the head the remainder of the way to its next microtubule binding site, then it seems possible that evidence for substeps may be very difficult to discover, in practice. That difficulty would be exacerbated if the distance subtended by the conformational component constituted a comparatively small fraction of the overall step (say, ∼2 nm, measured at the common stalk joining the heads) and the diffusional distance is larger. At least four single-molecule experiments bear directly on this question (16.Hua W. Chung J. Gelles J. Distinguishing inchworm and hand-over-hand processive kinesin movement by neck rotation measurements.Science. 2002; 295: 844-848Crossref PubMed Scopus (173) Google Scholar, 17.Asbury C.L. Fehr A.N. Block S.M. Kinesin moves by an asymmetric hand-over-hand mechanism.Science. 2003; 302: 2130-2134Crossref PubMed Scopus (442) Google Scholar, 18.Kaseda K. Higuchi H. Hirose K. Alternate fast and slow stepping of a heterodimeric kinesin molecule.Nat. Cell Biol. 2003; 5: 1079-1082Crossref PubMed Scopus (155) Google Scholar, 19.Yildiz A. Tomishige M. Vale R.D. Selvin P.R. Kinesin walks hand-over-hand.Science. 2004; 303: 676-678Crossref PubMed Scopus (756) Google Scholar). The Gelles lab (16.Hua W. Chung J. Gelles J. Distinguishing inchworm and hand-over-hand processive kinesin movement by neck rotation measurements.Science. 2002; 295: 844-848Crossref PubMed Scopus (173) Google Scholar) found that the short kinesin stalk of a recombinant Drosophila construct (K448 with a C-terminal biotinylation site) was torsionally rigid, a finding that contrasted sharply with earlier measurements of the stalk from full-length bovine kinesin, which was found to be surprisingly flexible overall, permitting kBT of energy to twist the stalk by more than one full rotation (20.Hunt A.J. Howard J. Kinesin swivels to permit microtubule movement in any direction.Proc. Natl. Acad. Sci. USA. 1993; 90: 11653-11657Crossref PubMed Scopus (103) Google Scholar). The rigidity of the short recombinant stalk allowed them to track the rotational Brownian motion of microtubules moved by single kinesin molecules. That movement was found to be tightly bounded, and did not produce large angular motions of 180° or more during stepping motion. In their article, Gelles and co-workers introduced important terminology for three different types of kinesin walk: symmetric hand-over-hand (where the two heads exchange leading and trailing positions on the microtubule, but the three-dimensional structure of the kinesin molecule is preserved at all equivalent points in the step cycle), asymmetric hand-over-hand (where the kinesin heads exchange positions on the microtubule, but the initial and final states of the molecule are not symmetry-related, implying that alternate steps must differ in essential ways), and inchworm (where one head always leads and the other always trails during the cycle of advancement; all inchworm models are necessarily symmetric). The failure to observe large angular changes in the stalk ruled out the symmetric hand-over-hand (HoH) model, which would have produced 180° stalk rotations. The body of evidence was therefore interpreted as favoring the inchworm model. However, as Hua et al. were careful to point out, the asymmetric HoH model could not be ruled out altogether by their data, although it would place severe constraints on the ways in which the molecule might move between stepping states. They wrote: “Thus, although our experimental results do not rigorously exclude an asymmetric hand-over-hand mechanism, we regard as improbable the existence of two structures that simultaneously satisfy all of the requirements outlined above.” The subsequent discovery of “limping” in kinesin, where the average kinetics of every other step switch between a faster and a slower stepping phase, proved that kinesin dimers advance through an asymmetric HoH motion, and that this motion is inconsistent with either the inchworm or symmetric HoH patterns. This is because kinesin dimers were found to alternate between two distinct (identifiable) states with each step, precisely as required by the asymmetric HoH model, which alone breaks symmetry: no such alternation can exist in either the (symmetric) inchworm or symmetric HoH models. Limping kinesins were generated in two rather distinct ways, using recombinant constructs of Drosophila kinesin. Work by Kaseda et al. (18.Kaseda K. Higuchi H. Hirose K. Alternate fast and slow stepping of a heterodimeric kinesin molecule.Nat. Cell Biol. 2003; 5: 1079-1082Crossref PubMed Scopus (155) Google Scholar) produced heterodimers with one “wild-type” head and the other head slowed by a mutation to the nucleotide binding pocket (R14A), which reduces the microtubule-stimulated ATPase rate by nearly 20-fold. Independent work by Asbury et al. (17.Asbury C.L. Fehr A.N. Block S.M. Kinesin moves by an asymmetric hand-over-hand mechanism.Science. 2003; 302: 2130-2134Crossref PubMed Scopus (442) Google Scholar) found that appropriate homodimer constructs of kinesin would also limp, provided that their stalk regions were sufficiently short. In fact, the degree of limping was found to be anti-correlated with the length of the stalk. Reports of kinesin limping were very soon followed by some compelling experiments from Paul Selvin’s group that followed the motion of an individual dimeric kinesin head labeled by a single fluorophore, using video centroid tracking accurate to nearly 1 nm (19.Yildiz A. Tomishige M. Vale R.D. Selvin P.R. Kinesin walks hand-over-hand.Science. 2004; 303: 676-678Crossref PubMed Scopus (756) Google Scholar). Kinesin heads (with labels on the heavy chain placed sufficiently close to the head domain) appeared to advance in a series of ∼16 nm steps, a result consistent with HoH motion but inconsistent with inchworm motion, which would have produced ∼8 nm steps instead. Importantly, however, and in contrast to the two earlier limping experiments, the centroid-tracking experiments do not distinguish between symmetric and asymmetric HoH motion, a fact that seems to have eluded more than one review writer. Modeling of biochemical kinetic results by Schief et al. (21.Schief W.R. Clark R.H. Crevenna A.H. Howard J. Inhibition of kinesin motility by ADP and phosphate supports a hand-over-hand mechanism.Proc. Natl. Acad. Sci. USA. 2004; 101: 1183-1188Crossref PubMed Scopus (96) Google Scholar) also supported HoH motions, as opposed to inchworming. Because the results of the Selvin lab support either symmetric or asymmetric HoH stepping models, whereas the results of the Gelles lab support either inchworm or asymmetric HoH models, the only stepping pattern consistent with both sets of results is asymmetric HoH motion. This, of course, is fully consistent with the two limping reports, which unambiguously indicated asymmetric HoH motion. All in all, the body of evidence in favor of the asymmetric HoH model is very compelling. We still don’t know what causes limping in homodimer constructs, but our experimental results suggest that it is unlikely to be simply an artifact of the linking geometry to the bead itself. Kinesin homodimers with short stalks limp whether bound to beads by streptavidin- or by antibody-based linkages. The degree of limping correlates with the length of the stalk and the value of the external load, and is most pronounced when the load is highest. This result is not consistent with some form of nonspecific interaction between one of the heads and the bead, an interaction that would be destabilized (and therefore diminished) at higher loads; this explanation therefore gives the wrong sign for the load-dependence. Moreover, if one head were to interact transiently with the bead for a significant portion of the cycle (as required for this explanation to hold), then the position of the bead would tend to report the position of a single head, rather than the centroid of the molecule (the stalk position), leading to alternating step sizes as well as step timing, contrary to observation. Dimers that are cross-linked by disulfide linkages between cysteines introduced into the proximal dimerization domain at the base of the stalk continue to limp, suggesting that helix misregistration of the coiled-coil region cannot be responsible for the phenomenon (Block lab, unpublished data). However, there are several other candidate explanations that are currently under test, and some of these involve torsional effects of the heads with respect to the stalk. Given the body of evidence in support of an asymmetric HoH stepping pattern, an obvious question arises as to how symmetry is actually broken for kinesin, which surely involves the microtubule itself. A corollary of the asymmetric HoH walk is that there must be two intrinsically different kinds of steps taken by kinesin molecules (call these a “left” step and a “right” step), and that these steps differ in both their trajectories (i.e., in the underlying molecular geometry) and also in their biochemical kinetics. Notwithstanding, the left and right kinesin steps are generated by head domains that are nominally identical in amino acid sequence (at least for homodimers), and the same head can generate either a left or a right step depending on its microenvironment. The consequences of this are far-reaching and profound, I believe. The temporal sequencing involved in stepping requires some form of communication between the heads to synchronize their biochemical cycles in precisely such a way as to maintain them out of phase, or else processivity would rapidly be lost. Furthermore, the evidence that kinesin’s 8-nm step is tightly coupled to the hydrolysis of a single ATP molecule (22.Hua W. Young E.C. Fleming M.L. Gelles J. Coupling of kinesin steps to ATP hydrolysis.Nature. 1997; 388: 390-393Crossref PubMed Scopus (281) Google Scholar, 23.Schnitzer M.J. Block S.M. Kinesin hydrolyses one ATP per 8-nm step.Nature. 1997; 388: 386-390Crossref PubMed Scopus (641) Google Scholar, 24.Coy D.L. Wagenbach M. Howard J. Kinesin takes one 8-nm step for each ATP that it hydrolyzes.J. Biol. Chem. 1999; 274: 3667-3671Crossref PubMed Scopus (300) Google Scholar) also implies some form of coordination between the cycles of the two heads. In fact, kinetic data on single-headed motors support the notion that processivity derives from head coordination (25.Berliner E. Young E.C. Anderson K. Mahtani H.K. Gelles J. Failure of a single-headed kinesin to track parallel to microtubule protofilaments.Nature. 1995; 373: 718-721Crossref PubMed Scopus (163) Google Scholar, 26.Hancock W.O. Howard J. Processivity of the motor protein kinesin requires two heads.J. Cell Biol. 1998; 140: 1395-1405Crossref PubMed Scopus (241) Google Scholar). The only realistic basis for such a gating mechanism would seem to be the mechanical strain that develops between heads during stepping itself. In principle, there are two plausible candidates for communicating this strain: through the regions joining the two kinesin heads, i.e., the neck linker regions and the common stalk, or from the heads through the microtubule protofilament. Of course, these are not mutually exclusive. Furthermore, whenever discussing the effects of strain on movement, one must remain mindful of the inherent reciprocity between the mechanics and the biochemistry: the load can affect the binding and hydrolysis, but binding and hydrolysis equally well affect the forces generated. These are intimately linked. Broadly speaking, two general classes of gating mechanism have been entertained. In one (the so-called “gated rear head” mechanism), the mechanical release of the trailing head from the microtubule leading head is accelerated by internal strain (27.Hancock W.O. Howard J. Kinesin’s processivity results from mechanical and chemical coordination between the ATP hydrolysis cycles of the two motor domains.Proc. Natl. Acad. Sci. USA. 1999; 96: 13147-13152Crossref PubMed Scopus (185) Google Scholar). Experimental support for this picture comes from the work of Crevel et al. (28.Crevel I.M. Nyitrai M. Alonso M.C. Weiss S. Geeves M.A. Cross R.A. What kinesin does at roadblocks: the coordination mechanism for molecular walking.EMBO J. 2004; 23: 23-32Crossref PubMed Scopus (73) Google Scholar) and Schief et al. (21.Schief W.R. Clark R.H. Crevenna A.H. Howard J. Inhibition of kinesin motility by ADP and phosphate supports a hand-over-hand mechanism.Proc. Natl. Acad. Sci. USA. 2004; 101: 1183-1188Crossref PubMed Scopus (96) Google Scholar), who reported that strain accelerates the detachment rate of the rear head. In the other model (the so-called “gated front head” mechanism), ATP binding to the leading head is suppressed through internal strain (29.Rosenfeld S.S. Fordyce P.M. Jefferson G.M. King P.H. Block S.M. Stepping and stretching. How kinesin uses internal strain to walk processively.J. Biol. Chem. 2003; 278: 18550-18556Crossref PubMed Scopus (158) Google Scholar, 30.Klumpp L.M. Hoenger A. Gilbert S.P. Kinesin’s second step.Proc. Natl. Acad. Sci. USA. 2004; 101: 3444-3449Crossref PubMed Scopus (96) Google Scholar). Note that these are not mutually exclusive, either, so mixed models are feasible. Work on head unbinding forces by the Ishiwata group has also helped to establish the notion that kinesin’s affinity for nucleotide is dependent on the directionality of an external load, and the apparent KD of a kinesin head for ADP is weakened up to sevenfold for rearward versus forward load (31.Uemura S. Ishiwata S. Loading direction regulates the affinity of ADP for kinesin.Nat. Struct. Biol. 2003; 10: 308-311Crossref PubMed Scopus (103) Google Scholar). Additional evidence supporting a gated front head mechanism comes from recent work by Guydosh and Block (8.Guydosh N.R. Block S.M. Backsteps induced by nucleotide analogs suggest the front head of kinesin is gated by strain.Proc. Natl. Acad. Sci. USA. 2006; 103: 8054-8059Crossref PubMed Scopus (109) Google Scholar) on the effects of nucleotide analogs (AMP-PNP and ADP·BeFx) on single-molecule motion driven by ATP. The addition of low concentrations of these nonhydrolyzable analogs causes stepping kinesin molecules to enter into long pauses, until the analogs can be released and ultimately exchanged for ATP. After a pause induced by an analog, it was discovered that processive stepping could only resume once the kinesin molecule took an obligatory, terminal backstep, exchanging the positions of its leading and trailing heads, which allows release of the bound analog from the (new) front head. Preferential release of the analog from the front head, as opposed to the rear head, implies that the kinetics of the two heads are differentially affected when both are bound to the microtubule. Kinesin, then, would seem to be the proverbial “back seat driver”, where the passenger head in the rear directs the driver head in the front! According to Hancock and Howard (27.Hancock W.O. Howard J. Kinesin’s processivity results from mechanical and chemical coordination between the ATP hydrolysis cycles of the two motor domains.Proc. Natl. Acad. Sci. USA. 1999; 96: 13147-13152Crossref PubMed Scopus (185) Google Scholar), release of stored strain upon unbinding of the trailing head permits the leading head to power an 8-nm advance of the entire molecule. According to Rice et al. (32.Rice S. Lin A.W. Safer D. Hart C.L. Naber N. Carragher B.O. Cain S.M. Pechatnikova E. Wilson-Kubalek E.M. Whittaker M. A structural change in the kinesin motor protein that drives motility.Nature. 1999; 402: 778-784Crossref PubMed Scopus (649) Google Scholar), ATP binding induces the docking of the neck linker on the leading head to produce motion of the partner head. My own group has found that the effective binding rate for ATP is load-dependent, which indicates that ATP binding, or a transition closely coupled to it, generates the forward step (33.Block S.M. Asbury C.L. Shaevitz J.W. Lang M.J. Probing the kinesin reaction cycle with a 2D optical force clamp.Proc. Natl. Acad. Sci. USA. 2003; 100: 2351-2356Crossref PubMed Scopus (250) Google Scholar). When taken together with other biochemical results, modeling of our data suggests that ATP binding is highly reversible and followed by some kind of conformational (and less reversible) change, leading to a mechanical step broadly consistent with the model of Rice et al. (32.Rice S. Lin A.W. Safer D. Hart C.L. Naber N. Carragher B.O. Cain S.M. Pechatnikova E. Wilson-Kubalek E.M. Whittaker M. A structural change in the kinesin motor protein that drives motility.Nature. 1999; 402: 778-784Crossref PubMed Scopus (649) Google Scholar). The recent finding by Guydosh and Block (8.Guydosh N.R. Block S.M. Backsteps induced by nucleotide analogs suggest the front head of kinesin is gated by strain.Proc. Natl. Acad. Sci. USA. 2006; 103: 8054-8059Crossref PubMed Scopus (109) Google Scholar) that the duration of the terminal backstep before the resumption of forward movement (from a pause induced by a nucleotide analog) depends on ATP concentration strengthens the case for a mechanical step triggered by ATP binding, and further argues against the alternative picture that the release of strain permits a step. Occasional backsteps have been reported since the very first studies of kinesin stepping under load (34.Svoboda K. Block S.M. Force and velocity measured for single kinesin molecules.Cell. 1994; 77: 773-784Abstract Full Text P
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