Tension-induced suppression of allosteric conformational changes explains coordinated stepping of kinesin-1

  1. Tsukasa Makino
  2. Ryo Kanada
  3. Teppei Mori
  4. Ken-ichi Miyazono
  5. Yuta Komori
  6. Haruaki Yanagisawa
  7. Shoji Takada
  8. Masaru Tanokura
  9. Masahide Kikkawa
  10. Michio Tomishige

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Abstract

The dimeric motor protein kinesin-1 walks along microtubules by alternating ATP hydrolysis and movement of its two motor domains (“head”). The detached head preferentially binds to the forward tubulin-binding site after ATP binds to the microtubule-bound head, but the mechanism preventing premature binding to the microtubule while the partner head awaits ATP remains unknown. Here, we examined the role of the neck linker, the segment connecting the two heads, in this mechanism. High-resolution structural analyses of the nucleotide-free head revealed a bulge just ahead of the neck linker’s base that creates an asymmetric constraint on its mobility. While the neck linker can stretch freely backward, it must navigate around this bulge to extend forward. Based on this finding, we hypothesized that premature binding of the tethered head is suppressed by an intolerable increase in neck linker tension. Molecular dynamic simulations and single-molecule fluorescent assays supported this model. These findings demonstrate a tension-based regulation mechanism where off-pathway conformational transitions are thermodynamically suppressed through entropy loss associated with neck linker stretching, suggesting that neck linker tension influences the allosteric conformational transition rather than directly affecting the nucleotide state.

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    Reply to the reviewers

    We are grateful to the reviewers for their detailed evaluation and insightful comments, which have improved the clarity and readability of this manuscript. We have addressed all reviewer comments and incorporated their suggested changes into the text and figures. The line numbers in our response correspond to those in the revised manuscript. Following reviewer 3’s comment, we have repeated the structural refinement of G234A and G234V apo crystal structures without water molecules, which improved the reliability of the data.

    Reviewer #1

    Abstract: The current abstract is challenging to follow. For instance, the phrase "The detached head preferentially binds to the forward tubulin-binding site after ATP binding, but the mechanism preventing premature binding to the microtubule while awaiting ATP remains unknown" could imply that the tethered head binds ATP, which is misleading. A clearer statement would be: "The detached head preferentially binds to the forward tubulin-binding site after ATP binding to the leading, microtubule-bound head, but the mechanism preventing premature binding to the microtubule while its partner awaits ATP remains unknown." Response: We thank the reviewer for the suggestion to improve clarity. We have revised the indicated sentence and updated the abstract to enhance clarity.

    Terminology: In the introduction, consider rephrasing to "...its two motor domains ("heads")."

    Response: We have corrected the phrase accordingly (line 44).

    Lines 71-72: The sentences "This mechanism explains how the tethered head preferentially binds to the forward-binding site 'after ATP binding.' However, it does not clarify how the tethered head is prevented from rebinding to the rear-tubulin binding site 'before ATP binding'" could be rephrased for clarity. A suggested revision is: "This mechanism explains how the tethered head preferentially binds to the forward-binding site after ATP binding to the microtubule-bound, leading head. However, it does not clarify how the tethered head is prevented from rebinding to the rear-tubulin binding site before ATP binds to the leading head."

    Response: We appreciate the suggestion for clarification. We have corrected the phrase accordingly (lines 72-75).

    Line 98: Consider revising "could release both ADP" to "could release both ADPs" or "could release both ADP molecules."

    Response: We have corrected the phrase accordingly (line 100).

    Lines 103-104: The statement "Therefore, these results suggest the tension posed to the neck linker plays a critical role in suppressing microtubule-binding of the tethered head" should be clarified. Since tension only develops in the two-heads-bound state, using "steric hindrance" instead of "tension" may improve precision.

    Response: We have corrected this sentence as follows: “These findings suggest that constraints on the neck linker (whether from steric hindrance or interactions with the head or microtubule) are crucial in preventing the tethered head from binding to microtubule” (lines 105-107).

    Lines 374-375: Replace "...before ATP-binding triggers the forward stepping..." with "...before ATP binding to the leading head triggers the forward stepping..."

    Response: We have corrected the phrase accordingly (line 374-375).

    Tense Consistency: Ensure consistent use of present or past tense throughout the manuscript for clarity.

    Response: We have reviewed the manuscript and corrected the verb tenses.

    Reviewer #2

    Lines 72-73 can be deleted as they are repetitive with lines 95-96. Response: While I acknowledge the reviewer’s point about redundancy, we would like to retain this sentence as it provides an important connection to the opening sentence of the next paragraph, where we explain why the rear-head gating model is required.

    Line 87: The authors should cite Mickolaczyk et al. PNAS 2015 and Sudhakar et al. Science 2021 as these studies also observed that the trailing head takes a sub-step and is located on the right side of the leading head before it moves forward and completes the step.

    Response: We did not cite these two papers as they contradict the statement of this sentence and rather suggest that kinesin waits for ATP-binding in the “two-head-bound” state. We interpreted this discrepancy as follows: 1) Mickolaczyk’s observations likely represent multiple motor-driven movement. Ensuring mono-valency of bead labeling is essential. In optical trapping assays, it is established that >98% of the bead motility is driven by a single motor when less than 50% of beads moved along the microtubule when brought into contact with microtubule using optical trap. The corresponding author has extensive experience preparing monovalent probes for optical trapping bead assays and high-speed single-molecule assays using gold probe (Tomishige et al., J. Cell Biol. 142, 989 (1998)), having established reliable protocols for monovalent labeling of kinesin with gold probes (refer to methods in Isojima et al., Nat. Chem. Biol. 2016 and Niitani et al. biorxiv 2024). The colloidal gold was coated with three SAMs (self-assembled monolayers) in a ratio of 1:10:10 (biotin-SAM:carboxy-SAM:hydroxy-SAM) to reduce surface biotin molecules and non-specific kinesin binding. The gold particles and kinesin-streptavidin complex were mixed at a 1:1 ratio, though this mixing ratio does not guarantee that 100% of the gold particle movements along microtubule are driven by single motors. We established that standard deviations (s.d.) of on- and off-axis displacements (especially that of off-axis) are key indicators for distinguishing between single- and multiple-motor driven motility of the gold probe. Under the above single-molecule conditions, majority of off-axis s.d. traces exhibited clear two-state transitions between microtubule-bound (low s.d.) and -unbound (high s.d.) states of the gold-labeled head, while under multivalent conditions (with higher kinesin:gold ratio and/or higher biotin-SAM ratio on the gold surface), most traces showed sub-steps but lacked these two-state transitions, instead displaying uncorrelated on- and off-axis s.d. traces. In contrast, Mickolajczyk et al. used commercial streptavidin-coated gold nanoparticles mixed with kinesin at a 6:1 motor-to-gold ratio. While their 2016 and 2017 papers did not show s.d. traces, their Biophys. J. 2019 paper (Fig.4) displayed s.d. traces that are characteristic of multivalent bead motility according to the criteria described above. 2) Sudhakar et al.’s interpretation that rapid sub-steps between 8-nms steps represent tethered head movement (illustrated in Fig 4 of their paper) is likely incorrect. The optical trap force acts on the neck linker of the microtubule-bound head, not to the neck linker of the tethered head. Consequently, trailing head detachment should not cause significant displacement of the trapped bead (as illustrated in Fig. 4 of Carter and Cross, Nature 2005). Instead, conformational changes in the neck linker of the microtubule-bound head (i.e., cover-neck bundle formation after ATP binding (Hwang et al. Structure 2008)) would cause bead displacement, supporting that kinesin waits for ATP in the “one-head-bound state”.

    Lines 103: The authors should cite Benoit et al. kinesin14 and Kif1A structures as these studies directly show the conformations of the neck-linkers when both heads are bound to the microtubule.

    Response: We cited the paper (line 105).

    Line 113: There is an extra "e" on "nucleotide".

    Response: We have corrected the typo (line 117).

    Line 118: I would delete "universal" as it is not clear whether all kinesins use a tension-based mechanism.

    Response: We agree with the reviewer’s comment. Further, reviewer 3 noted that recent studies showed that kinesin-3 may not be explained by this mechanism, so we have removed the word “universal” from this sentence as well as from the Abstract and Discussion.

    Line 132: Why did the authors decide to use a cys-lite mutant for X-ray and cryo-EM studies?

    Response: We used the Cys-light mutant to maintain consistency across various experimental techniques in this paper and to enable direct comparison with the nucleotide-free kinesin-1 structures reported by Cao et al. (2014, 2017), who used the same Cys-light construct. To express this, we revised the sentence as follows: “For consistency across experimental techniques and comparison with the previously solved nucleotide-free kinesin-1 structures, we used a cysteine-light mutant kinesin, where surface-exposed cysteines were replaced with either Ala or Ser” (lines 135-138).

    Line 192: The authors refer to Figures 3A and B when they discuss ATP-like and ADP-like conformations. However, these figures refer to open, semi-open, and closed conformations. Things become clear later in the text, but this is confusing, as is. I recommend the authors either show ATP-like and ADP-like classification as a supplemental figure and refer to that figure or not refer to the figure in this sentence.

    Response: To explain the result in this paragraph, we should reference these figures, while we acknowledge the reviewer’s comment about the confusing nomenclature in Fig.3. To address this, Fig. 3A now lists both the old terminology (nucleotide-free, ADP-like, and ATP-like) alongside the new terminology (open, semi-open, and closed).

    Lines 259-260: I would delete "as evidenced by..." and just cite those papers.

    Response: We have corrected this sentence accordingly (line 265-266).

    Lines 262-276: The authors should cite the relevant literature in this paragraph as most of their conclusions here were already shown by previous structural studies.

    Response: Reviewer 3 also noted that this paragraph outlines our current understanding, which seems out of place in the Results and more relevant for the Discussion. Therefore, we have moved this paragraph to the Discussion section and added relevant citations from the literature (lines 390-406).

    Recent biophysical studies claim that neck-linker docking is a two-step process that occurs in ATP binding and ATP hydrolysis. Do the authors agree with this model? Can they comment on why the neck-linker only partially docks during ATP binding, and require ATP hydrolysis to complete the docking? If they disagree with this model, this should be explained in the Discussion.

    Response: This paper focuses on the neck linker’s extensibility in coordinated motility rather than its docking onto the head. The correlation between ATP binding/hydrolysis and neck linker-docking has been examined in a concurrent paper by Niitani et al. (biorxiv 10.1101/2024.09.19.613828) and is discussed in their Discussion section. In this paper, using loose backward constraint on the neck linker, we demonstrated that docking of the initial neck linker segment is sufficient to half-open the gate. Furthermore, extending the neck linker length increased the ATP off-rate of the rear E236A head, indicating that forward neck linker strain plays a crucial role in stabilizing the closed state. These findings support the hypothesis that neck linker docking remains partially unstable in the one-head-bound state and achieves full stabilization only after transitioning to the two-head-bound state.

    Lines 285: The authors should cite Benoit et al. as they showed this clearly in their structure. Benoit et al. showed that, even though both heads are bound to AMP-PNP, the neck linkers are pointed in opposite directions and the rigid body conformations of the trailing and leading heads are different. Do the authors take this into account when they model the Topen-Lopen state? Can they also comment on why the heads can have different rigid body conformations even though they are bound to the same nucleotide? Is this because tension on the neck-linker is too high if both heads are in the open conformation?

    Response: We have added a citation to Benoit et al. 2021. The Topen-Lopen state is an off-pathway conformational state that differs from the on-pathway two-head-bound states (Tclosed-Lopen) studied using cryoEM. Using smFRET, we showed this state appeared only in the neck linker extended mutants, for which no cryoEM observation exist. Therefore, we modeled the Topen-Lopen state by assuming both heads adopt identical conformations in the open state, and showed that this off-pathway transition is suppressed because it would cause an intolerable increase in neck linker tension. Benoit et al.’s finding that the front open head can bind AMPPNP aligns with Niitani et al.’s observation (bioRxiv 2024) that while the front head can bind ATP, it maintains a low ATP affinity state—unlike the rear head, which exhibits high ATP affinity. This suggests that ATP binding (nucleotide state) is not tightly coupled to the open-to-closed conformational transition of the head.

    Line 308: How do the authors estimate the tension on the neck linker? This needs to be explained briefly in the main text as it is central to the conclusions of this work.

    Response: While we briefly described the method to estimate the tension in the text, we did not specify which part of the disordered neck linker was used for this calculation. We have now added this explanation as follows: “To estimate the amount of this tension, we isolated the disordered neck linker segments from both the leading and trailing heads that are stretched between the motor domains without steric hindrance or docking onto the head (Fig. S4 D). Then, we applied a harmonic potential to the Cα atoms at both ends of the stretched region and calculated the tension from the average displacement of the Cα atom from the potential minimum using MD simulations (Fig. 7, A and B)” (lines 300-306)

    Line 308: Calculated tension is a lot higher than the force needed to pull a tubulin out from its tail from the microtubule (Kuo et al. Nat Comms 2022). Even the lowest tension they reported is a lot higher than the estimates made by Clancy et al. and Hyeon and Onuchic. The authors should comment on why this might be the case.

    Response: The neck linker tension between two heads differs from the force applied by the optical trap to the bead attached to the coiled-coil stalk. Because these forces act in different direction and the coiled-coil stalk contains flexible hinges, torques, rather than forces, should be compared, though this is difficult to estimate (as described in Figure S16 in Hwang and Karplus, Structure 16, 62-71 (2008)). Hyeon & Onuchi (2007) and Hariharan & Hancock (2009) calculated the neck linker tension using a worm-like chain model, yielding different results of 12-15 pN and 28 pN, respectively (Clancy et al. cited these results). This discrepancy stems from different end-to-end distances used in their calculations (3.1 nm versus 4 nm). The 4 nm distance used by Hariharan and Hancock likely represents the tension in the two-head-bound state, as it equals half the distance between two heads on adjacent tubulin-binding sites. Using MD simulation, Hariharan and Hancock further estimated the neck linker tension of 15 pN in constraint force mode and 35 pN in force-clamp mode. Our estimated tension (39 pN) in Tclosed-Lopen state is comparable to the upper limit of these calculations. This estimated tension using isolated neck linkers is likely an overestimate, since the stretched neck linker in the presence of the motor domain includes an additional energetic contribution from its direct interaction with the leading head, which will be described in detail in our response to the reviewer 2’s comment #16. To address this, we have included the following sentence: “The tension in the Tclosed-Lopen state is likely an overestimate since this measurement excludes the enthalpic component discussed above, though it is comparable to previous MD measurements and theoretical calculations using a worm-like chain model (Hariharan and Hancock, 2009).” (lines 307-311)

    Line 321: I would also cite Shastry and Hancock here.

    Response: We have cited this paper (line 322).

    Lines 387: "...the transition from one-head-bound to two-head-bound Topen-Lopen state".

    Response: We have corrected the phrase accordingly (lines 387-388).

    Lines 418-428: The authors assume that the neck-linker extension is purely entropic. However, neck linkers are almost fully stretched especially in unfavorable two-head-bound conformations, and they can potentially make contact with the motor domains. Therefore, this process may not be purely entropic and may also involve energetic terms when considering the free energy of neck linker docking.

    Response: We appreciate the reviewer’s comment, as we had overlooked this important point. After examining the simulation movies of neck linker dynamics in Topen-Lopen and Tclosed-Lopen states (Fig. S4B, C and Videos 3, 4), we found that the stretched neck linker region in the Topen-Lopen state was displaced from the head and showed no interaction with the head during the simulation period. However, in the Tclosed-Lopen state, we observed a stable interaction between the K326 residue in the neck linker and the D37 and F48 residues of the leading open head (which can be seen in Video 4). This interaction was not included in our tension estimation (Fig. S4D), which assumed the tension had a purely entropic origin. Therefore, the estimated tension in the Tclosed-Lopen state is likely an overestimate, while the tension in the Topen-Lopen state remains purely entropic. We have added two sentences to describe these observations as follows: “Throughout the simulation, the stretched neck linker remained displaced from the head without any interaction, suggesting that the neck linker behaves as an entropic spring.” (lines 288-290), and “During this simulation, we observed a stable contact between the K326 side chain of the disordered neck linker and the D37 and F48 residues of the leading head (see Video 4), suggesting that the neck linker tension in Tclose-Lopen state includes an energetic component.” (lines 293-296)

    Lines 452-454: I think this sentence summarizes the most significant contribution of this work and should be clearly mentioned in the abstract.

    Response: We thank the reviewer for this suggestion and have incorporated the sentence into the abstract.

    Lines 476-479: This sentence claims that neck linker docking is not necessary. Instead, rotation of the R-sub domain of the motor domain is sufficient to trigger the forward step. I would omit this sentence, as the rationale is not well explained, and it conflicts with a large body of literature on neck-linker docking. This could be an interesting idea to discuss in a perspective article or a topic of future research, but it may unnecessarily confuse the reader at the conclusion of this work.

    Response: We included this sentence because it provides a testable prediction for neck linker-docking independent stepping, and we are preparing a manuscript to experimentally test this hypothesis. However, we agree with the reviewer’s comment that this statement conflicts with the common view in this field, and without additional verification or statement, it would confuse readers. Therefore, we have removed this sentence from the manuscript.

    Reviewer #3

    Major Comments:

    1. The Abstract is not clearly written to distinguish which kinesin head is being discussed.

    Response: We revised the second sentence in the abstract to distinguish between the tethered and microtubule-bound heads and updated the abstract to enhance clarity.

    The authors describe the bulge formed by the terminus if helix 4 as an obstruction that is "creating an intolerable increase in neck linker tension", but could it not simply be that forward head binding is conformationally disfavoured? Perhaps these ideas are not mutually exclusive.

    Response: We agree with the reviewer that in the ATP-waiting state, the tethered head might also be prevented from binding to the tubulin-binding site due to the neck linker requiring a highly stretched configuration—this could occur before the tension increase that accompanies the transition from semi-open to open conformation. While we addressed this possibility in the Discussion section (lines 398-405 of the original version), our explanation was not sufficiently clear. We have therefore revised the sentence to clarify this point as follows: “Therefore, we can only speculate that the tension would lie somewhere between that of the Tclose-Lopen and Topen-Lopen states, and that microtubule binding of the tethered semi-open head may be restricted because the disordered neck linkers would need to adopt highly stretched configurations.” (lines 421-424)

    The term "universal" in describing this tension-based regulation mechanism seems unjustified without examination of other kinesins. They might consider Kif1A as a subject given its shorter and seemingly more entropically-constrained neck linker. Recent structures of Kif1A bound to MTs in two-heads bound states have recently been described by Benoit et al. (Nat Comm. 2024).

    Response: We agree with the reviewer and acknowledge that this tension-based regulation mechanism may not apply to some other kinesin subfamilies, which have different neck linker properties, such as varying neck linker lengths or specific interactions with the motor domain. We removed the word “universal” from the Abstract, Introduction and Discussion and added a final sentence to the Discussion as follows: “Additionally, studies are needed to examine whether this mechanism extends to other kinesin subfamilies with different neck linker properties, such as varying neck linker lengths (kinesin-2: Hariharan and Hancock, 2009; kinesin-3: Benoit et al., 2024) or specific interactions with the motor domain (kinesin-6: Guan et al., 2016; Ranaivoson et al., 2023).” (lines 501-505).

    The authors should consider discussing how having two chains in the asymmetric unit of the APO motor impacts the NL structure.

    Response: The G234A apo and G234V apo crystals share the same asymmetric unit since the G234A crystal was grown from a G234V crystal seed. We inspected the structures near the proximal end of the neck linker (or the C-terminus of the a6 helix connected to neck linker) that could cause steric hindrance or direct interaction with the initial segment of the neck linker. The closest element of the adjacent chain was L5, which was separated by 1.1 nm from the proximal end of the neck linker (K324 residue) and did not interact with it. The proximal ends of the neck linkers of chains A and B face each other, with a cylindrical cavity between them. This cavity in G234V apo allows an antiparallel β-sheet formation between the two stretched neck linkers of chain A and B (Figure S2A). However, we did not observe density corresponding to the antiparallel β-sheet in the cavity of G234A apo, likely due to its slightly smaller cavity size. Notably, this antiparallel β-sheet formation would be geometrically impossible for the two neck linkers in a dimer since their C-termini are joined in parallel by the neck coiled-coil. These explanations have been added to the text (lines 154-156) and the legend of Figure S2.

    At barely 3 angstroms, how are waters modelled and how is it their B-factors are so low? Rfact and Rfree are also quite divergent for the GA mutant (APO) structure.

    Response: To improve the R-factor, we placed water molecules to account for unmodeled and discontinuous electron density peaks that were too small to be interpreted as polypeptides. However, this treatment was likely incorrect and is the primary reason for both the low B-factor and Rfree values, which led to the large discrepancy between Rwork and Rfree. To address this issue, we repeated the structural refinement of G234A and G234V apo structures by removing water molecules placed on unmodeled density peaks. We retained only one water molecule in the nucleotide pocket of chain A in the G234A apo structure due to its well-defined density (Figure S1). This improved refinement significantly reduced the discrepancy between Rwork and Rfree of G234A apo from 20.0/28.1% to 20.7/26.5%. For G234V apo, while the discrepancy remained unchanged, the overall values were improved from 24.4/29.2% to 20.0/25.8%. We updated Table 1 and deposited these refined structures to the Protein Data Bank (PDB# 9L78 and 9L6K) with details provided in the “Data availability” section.

    Lines 262-276: This section describes our current understanding of the mechanism of neck linker docking in accord with NP closure, which seems out of place in the Results and more relevant for the Discussion. Likewise, the two paragraphs before and after the description of the gold nanocluster study describe a re-evaluation and graphical/animated description of others' findings (Figure 4 and videos 1 and 2), rather than analysis of structural data obtained experimentally in this study.

    Response: We acknowledge that this paragraph describes previous findings rather than current results. Therefore, we have relocated it to the Discussion section with appropriate citations from the literatures (lines 390-406). In addition, the paragraph, which precedes the gold nanocluster study, draws from previous research using different subdomain boundaries, so we added the relevant citations accordingly (line 238).

    It is mentioned in the Discussion that the neck linker-docking is not necessary to trigger the forward step after ATP binding, but rather the rotation of the R-domain is sufficient to diminish the steric hindrance that limits tethered head binding. Are they suggesting that the neck linker could be undocked or disordered when making the forward step of a two-headed motor? According to other structural studies, a fully docked neck-linker is required to adopt the closed conformation. Moreover, binding of the leading head to the MT is necessary for complete closure of the nucleotide-binding pocket of the trailing head.

    Response: This sentence was included because it offers a testable prediction for neck linker-docking independent stepping, and we are currently preparing a manuscript to test this hypothesis experimentally. The prediction is supported by Niitani et al.’s finding (biorxiv 10.1101/2024.09.19.613828) that loose neck linker crosslinking, which allows docking of the initial segment of the neck linker onto the head but prevents complete neck docking, reduced ATP-induced microtubule detachment rate by half. However, since this statement challenges the conventional understanding in this field and requires further verification, as noted by reviewer 2, we have removed it to avoid confusion.

    Minor Comments:

    Line 113 - "nucletodiee-free" spelling.

    Response: We have corrected the typo (line 117).

    Lines 118-122 - Final sentence of Introduction needs improvement: "Moderate neck-linker extension"? Terms are not defined/vague.

    Response: To clarify this point, we revised this sentence as follows: “among possible conformational transitions, the one that requires less entropy reduction from stretching the disordered neck linker is favored” (lines 123-125).

    Line 131 - Possible Error: "N-terminal motor domain (1-332 residues)" - should this be 1-322?

    Response: This is our mistakes and we corrected the number of residues (line 134).

    It could be difficult for some readers to follow the naming convention used Tapo-Lapo which is equivalent to Topen-Lopen in the final mechanistic model figure.

    Response: In response to the reviewer’s comment, we have removed the reference to the Tapo-Lapo state from the Introduction and revised the notation in the Result section from Tapo-Lapo to Topen-Lopen.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    The manuscript by Makino et al. investigates the mechanistic basis by which the two motor domains (heads) of kinesin dimers coordinate binding and release from their microtubule (MT) trackway in a productive manner for motility (i.e., in a way that limits backsteps or abandoned steps and encourages directional movement).

    Earlier studies have provided structural, biochemical, and indirect visual evidence that kinesin dimers first associate with MTs with one head. This MT interaction opens the head's nucleotide pocket so its bound ADP can be released. ATP can then enter, and the nucleotide pocket will close around it when the neck linker at the C-terminus of the motor domain is able to dock against the side of the motor domain. The docking of the neck linker directs the tethered motor domain forward to the next available binding site on the MT, but before this happens, it is possible that the tethered head can still engage the MT, either in front of, or behind, the MT-bound head. Similarly, after taking a step, a head that has disengaged from the MT could rebind its previous site, or swing ahead of its partner motor domain to engage the next binding site on the MT.

    This paper used structural methods, computational modelling, molecular dynamics, and biophysical measurements of labeled mutant kinesin dimers to understand how these tethered head-MT interactions are restricted from happening until the other MT-bound head is in the correct catalytic state for the tethered head-MT interaction to be productive. Their goal was to understand the mechanism that prevents premature binding of the tethered head to the microtubule during ATP-waiting state.

    Their X-ray crystallographic and cryo-EM structures of monomeric kinesin-1 heads that were mutated to facilitate capture of the APO or "Open" nucleotide pocket state showed that the kinesin neck linker doesn't interact specifically or stably with either the motor domain or the microtubule surface in the nucleotide-free state. It appears that the neck linker is inhibited from docking and extending toward the MT plus end by a bulge made by the end of helix 4. This bulge would increase the distance the neck linker would have to stretch if it were connected to the neck linker of its MT-bound partner head. Thus, they proposed that this bulge deters kinesin dimers from being able to form complexes with MTs in which both the forward and rearward head are both bound to the MT and contain empty nucleotide pockets (i.e., both heads are in the 'open' state). Tension on the normal-length neck must therefore restrict unproductive MT binding events.

    Overall, this study makes interesting links between the asymmetries in neck linker tension, entropy levels, and nucleotide pocket status of each dimeric kinesin head.

    Major Comments:

    1. The Abstract is not clearly written to distinguish which kinesin head is being discussed.
    2. The authors describe the bulge formed by the terminus if helix 4 as an obstruction that is "creating an intolerable increase in neck linker tension", but could it not simply be that forward head binding is conformationally disfavoured? Perhaps these ideas are not mutually exclusive.
    3. The term "universal" in describing this tension-based regulation mechanism seems unjustified without examination of other kinesins. They might consider Kif1A as a subject given its shorter and seemingly more entropically-constrained neck linker. Recent structures of Kif1A bound to MTs in two-heads bound states have recently been described by Benoit et al. (Nat Comm. 2024).
    4. The authors should consider discussing how having two chains in the asymmetric unit of the APO motor impacts the NL structure.
    5. At barely 3 angstroms, how are waters modelled and how is it their B-factors are so low? Rfact and Rfree are also quite divergent for the GA mutant (APO) structure.
    6. Lines 262-276: This section describes our current understanding of the mechanism of neck linker docking in accord with NP closure, which seems out of place in the Results and more relevant for the Discussion. Likewise, the two paragraphs before and after the description of the gold nanocluster study describe a re-evaluation and graphical/animated description of others' findings (Figure 4 and videos 1 and 2), rather than analysis of structural data obtained experimentally in this study.
    7. It is mentioned in the Discussion that the neck linker-docking is not necessary to trigger the forward step after ATP binding, but rather the rotation of the R-domain is sufficient to diminish the steric hindrance that limits tethered head binding. Are they suggesting that the neck linker could be undocked or disordered when making the forward step of a two-headed motor? According to other structural studies, a fully docked neck-linker is required to adopt the closed conformation. Moreover, binding of the leading head to the MT is necessary for complete closure of the nucleotide-binding pocket of the trailing head.

    Minor Comments:

    Line 113 - "nucletodiee-free" spelling.

    Lines 118-122 - Final sentence of Introduction needs improvement: "Moderate neck-linker extension"? Terms are not defined/vague.

    Line 131 - Possible Error: "N-terminal motor domain (1-332 residues)" - should this be 1-322?

    It could be difficult for some readers to follow the naming convention used Tapo-Lapo which is equivalent to Topen-Lopen in the final mechanistic model figure.

    Significance

    The manuscript by Makino et al. explores the coordination of kinesin dimer motor domains during microtubule (MT) motility, focusing on the mechanism that prevents premature tethered head binding in the ATP-waiting state. The combination of structural biology (X-ray crystallography, cryo-EM), computational modeling, molecular dynamics, and biophysical studies on mutant kinesins is a strength of the study and has allowed the authors to provide insights into how neck linker tension, nucleotide pocket status, and structural features like a helix 4 bulge influence kinesin dynamics.

    Strengths

    1. The identification of the helix 4 bulge as a determinant of neck linker tension adds to our understanding of kinesin head coordination and motility.
    2. The study draws interesting links between entropy, structural asymmetries, and functional outcomes in kinesin dimer motility.
    3. The findings hint at conserved mechanisms regulating kinesin family motor dynamics, although this remains to be experimentally confirmed.

    Limitations

    1. Claims of universal applicability for the tension-based regulation mechanism are premature without examining other kinesins, such as Kif1A.
    2. The role of neck linker docking in forward stepping and the potential for undocked states during motility need clearer resolution against prior studies.

    In conclusion, the study contributes valuable mechanistic insights into kinesin motility and raises intriguing questions about its broader applicability across kinesin families, warranting further investigation. This study should be of general interest to the cytoskeletal motors community.

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    Referee #2

    Evidence, reproducibility and clarity

    This manuscript investigates the role of the neck linker in coordinating the stepping cycles of the two heads of a kinesin-1 motor.

    Previous studies in the field showed that kinesin walks by alternating stepping of its heads, referred to as hand-over-hand. In solution, both heads are in the ADP-bound state and have low affinity for MTs. One of the heads collides with the microtubule and releases its ADP while the other head remains in the ADP-bound state and does not interact with the microtubule. ATP binding to the bound head results in partial docking of its neck linker, which pulls the unbound head by 8.2 nm towards the plus end. ATP hydrolysis in the bound head completes its neck-linker docking and pulls the unbound head further towards the tubulin adjacent to the plus end side of the bound head. In this state, both heads are bound to the microtubule, the trailing head is bound to ADP.Pi and the leading head is nucleotide-free. ATP binding to the leading head is gated until the trailing head releases Pi and dissociates from the microtubule. After microtubule release, the head remains in the trailing position near its tubulin binding site as kinesin-1 waits for ATP binding to the leading head to start the next ATPase cycle.

    The authors of this study ask an important question: After the trailing head releases from the microtubule, what prevents it from binding the tubulin on the minus-end or plus-end side of the leading head as the motor waits for ATP binding to the leading head? They first obtained the crystal structure of the kinesin head plus its neck linker in the nucleotide-free and ADP-bound conditions. Next, they solved the microtubule-bound structure of a kinesin head in a nucleotide-free condition using cryo-EM. Using their structures and previous structural studies of kinesin motors, they discovered that rigid body motions within the kinesin motor domain upon ADP release result in a steric clash between the C-terminus of helix4 and the distal end of helix 6 where the neck-linker is connected. They claim that this steric clash imposes an asymmetric constraint on the mobility of the neck linker: it can stretch freely backward but not forward in this state. They supported their model by labeling the middle of the neck linker with a gold nanoparticle and finding its position relative to the motor domain bound to the microtubule using cryo-EM. They observed that the gold density is positioned backward and located on the right-hand side of the motor domain, providing an explanation for why the trailing head takes steps from the right side of the leading head as kinesin walks. Consistent with previous work, they showed that ATP binding to the head releases this constraint, the first two residues of the neck-linker extend helix 6, while the rest docks onto a hydrophobic pocket on the motor domain and forms a beta-sheet with the neck cover strand, completing the neck-linker docking. Towards the conclusion of this work, the authors built a model for the two-head-bound state of kinesin on the microtubule and calculated the tension on the neck linkers based on the rigid body conformations of the motor domains. Using MD simulations, they estimated that the heads experience 50-100 pN tension through the extension of their neck linkers to support both heads to bind to the microtubule. The tension is lowest when the trailing head is ATP-bound and the leading head is nucleotide-free (which is the estimated state of kinesin right after neck-linker docking and the forward stepping of the trailing head), whereas tension is prohibitively too high when both heads are in the nucleotide-free state or the trailing head is in the nucleotide-free state and the leading head is in the ATP bound state. These results are consistent with a large body of work in literature and suggest that tension on the linkers prevents rebinding of the trailing head to the microtubule, keeps the two heads out of phase, and coordinates the stepping cycle of the kinesin heads to proceed in the forward direction, rather than backward. Finally, they perform smFRET measurements on kinesin mutants with extended neck linkers and show that extension of the neck linkers allows both heads of a kinesin dimer to simultaneously bind to the microtubule, demonstrating that it is the tension that prohibits the trailing head from binding to the microtubule in the ATP waiting state and keeps kinesin in a one-head-bound state for the majority of its mechanochemical cycle.

    I only have several suggestions to improve the clarity and more balanced citation of the previous literature.

    1. Lines 72-73 can be deleted as they are repetitive with lines 95-96.
    2. Line 87: The authors should cite Mickolaczyk et al. PNAS 2015 and Sudhakar et al. Science 2021 as these studies also observed that the trailing head takes a sub-step and is located on the right side of the leading head before it moves forward and completes the step.
    3. Lines 103: The authors should cite Benoit et al. kinesin14 and Kif1A structures as these studies directly show the conformations of the neck-linkers when both heads are bound to the microtubule.
    4. Line 113: There is an extra "e" on "nucleotide".
    5. Line 118: I would delete "universal" as it is not clear whether all kinesins use a tension-based mechanism.
    6. Line 132: Why did the authors decide to use a cys-lite mutant for X-ray and cryo-EM studies?
    7. Line 192: The authors refer to Figures 3A and B when they discuss ATP-like and ADP-like conformations. However, these figures refer to open, semi-open, and closed conformations. Things become clear later in the text, but this is confusing, as is. I recommend the authors either show ATP-like and ADP-like classification as a supplemental figure and refer to that figure or not refer to the figure in this sentence.
    8. Lines 259-260: I would delete "as evidenced by..." and just cite those papers.
    9. Lines 262-276: The authors should cite the relevant literature in this paragraph as most of their conclusions here were already shown by previous structural studies.
    10. Recent biophysical studies claim that neck-linker docking is a two-step process that occurs in ATP binding and ATP hydrolysis. Do the authors agree with this model? Can they comment on why the neck-linker only partially docks during ATP binding, and require ATP hydrolysis to complete the docking? If they disagree with this model, this should be explained in the Discussion.
    11. Lines 285: The authors should cite Benoit et al. as they showed this clearly in their structure. Benoit et al. showed that, even though both heads are bound to AMP-PNP, the neck linkers are pointed in opposite directions and the rigid body conformations of the trailing and leading heads are different. Do the authors take this into account when they model the Topen-Lopen state? Can they also comment on why the heads can have different rigid body conformations even though they are bound to the same nucleotide? Is this because tension on the neck-linker is too high if both heads are in the open conformation?
    12. Line 308: How do the authors estimate the tension on the neck linker? This needs to be explained briefly in the main text as it is central to the conclusions of this work.
    13. Line 308: Calculated tension is a lot higher than the force needed to pull a tubulin out from its tail from the microtubule (Kuo et al. Nat Comms 2022). Even the lowest tension they reported is a lot higher than the estimates made by Clancy et al. and Hyeon and Onuchic. The authors should comment on why this might be the case.
    14. Line 321: I would also cite Shastry and Hancock here.
    15. Lines 387: "...the transition from one-head-bound to two-head-bound Topen-Lopen state".
    16. Lines 418-428: The authors assume that the neck-linker extension is purely entropic. However, neck linkers are almost fully stretched especially in unfavorable two-head-bound conformations, and they can potentially make contact with the motor domains. Therefore, this process may not be purely entropic and may also involve energetic terms when considering the free energy of neck linker docking.
    17. Lines 452-454: I think this sentence summarizes the most significant contribution of this work and should be clearly mentioned in the abstract.
    18. Lines 476-479: This sentence claims that neck linker docking is not necessary. Instead, rotation of the R-sub domain of the motor domain is sufficient to trigger the forward step. I would omit this sentence, as the rationale is not well explained, and it conflicts with a large body of literature on neck-linker docking. This could be an interesting idea to discuss in a perspective article or a topic of future research, but it may unnecessarily confuse the reader at the conclusion of this work.

    Significance

    Overall, this work is highly interesting and valuable to the kinesin field. It addresses an important question about the role of neck-linkers in the kinesin mechanism and provides meaningful explanations for some fo the previous observations made in the field.

    Expertise: I am a single-molecule biophysicist interested in the mechanism and regulation of microtubule motors.

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    Referee #1

    Evidence, reproducibility and clarity

    In this study, Makino et al. investigate how tension within the neck linker regulates the coordinated stepping of kinesin-1, a dimeric motor protein with two motor domains (or "heads"). Using high-resolution structural analyses, the authors identify a bulge near the neck linker's base in the nucleotide-free head that restricts forward extension, increasing steric hindrance when extended forward. This hindrance, they propose, prevents the tethered head from prematurely binding to the microtubule while the leading, microtubule-bound head awaits ATP. Molecular dynamic simulations and single-molecule fluorescence assays support this steric hindrance-based model, suggesting a mechanism that thermodynamically suppresses off-pathway transitions, thereby guiding kinesin-1's processive movement along microtubules. I recommend acceptance of the manuscript, subject to the following revisions:

    1. Abstract: The current abstract is challenging to follow. For instance, the phrase "The detached head preferentially binds to the forward tubulin-binding site after ATP binding, but the mechanism preventing premature binding to the microtubule while awaiting ATP remains unknown" could imply that the tethered head binds ATP, which is misleading. A clearer statement would be: "The detached head preferentially binds to the forward tubulin-binding site after ATP binding to the leading, microtubule-bound head, but the mechanism preventing premature binding to the microtubule while its partner awaits ATP remains unknown."
    2. Terminology: In the introduction, consider rephrasing to "...its two motor domains ("heads")."
    3. Lines 71-72: The sentences "This mechanism explains how the tethered head preferentially binds to the forward-binding site 'after ATP binding.' However, it does not clarify how the tethered head is prevented from rebinding to the rear-tubulin binding site 'before ATP binding'" could be rephrased for clarity. A suggested revision is: "This mechanism explains how the tethered head preferentially binds to the forward-binding site after ATP binding to the microtubule-bound, leading head. However, it does not clarify how the tethered head is prevented from rebinding to the rear-tubulin binding site before ATP binds to the leading head."
    4. Line 98: Consider revising "could release both ADP" to "could release both ADPs" or "could release both ADP molecules."
    5. Lines 103-104: The statement "Therefore, these results suggest the tension posed to the neck linker plays a critical role in suppressing microtubule-binding of the tethered head" should be clarified. Since tension only develops in the two-heads-bound state, using "steric hindrance" instead of "tension" may improve precision.
    6. Lines 374-375: Replace "...before ATP-binding triggers the forward stepping..." with "...before ATP binding to the leading head triggers the forward stepping..."
    7. Tense Consistency: Ensure consistent use of present or past tense throughout the manuscript for clarity.

    Significance

    The conclusions are supported by the data provided, offering valuable insights into the coordination of kinesin's motor domains during movement. These findings help address a knowledge gap in kinesin stepping mechanics, making this work relevant to researchers studying cytoskeletal motor proteins.

  5. Version published to 10.1101/2024.09.19.613825v2 on bioRxiv
  6. Version published to 10.1101/2024.09.19.613825v1 on bioRxiv