Structural model of microtubule dynamics inhibition by kinesin-4 from the crystal structure of KLP-12 –tubulin complex

  1. Shinya Taguchi
  2. Juri Nakano
  3. Tsuyoshi Imasaki
  4. Tomoki Kita
  5. Yumiko Saijo-Hamano
  6. Naoki Sakai
  7. Hideki Shigematsu
  8. Hiromichi Okuma
  9. Takahiro Shimizu
  10. Eriko Nitta
  11. Satoshi Kikkawa
  12. Satoshi Mizobuchi
  13. Shinsuke Niwa
  14. Ryo Nitta

  • Curated by eLife

    eLife logo

    Evaluation Summary:

    Here, Taguchi et al. study a member of the kinesin-4 family of motors, which is important in controlling microtubule length during normal development and maintenance. The authors aim to determine how a member of the kinesin-4 family is able to stabilize the tips of microtubules to suppress both their growth and shrinkage. This paper provides compelling data on KLP-12 by combining in vivo C. elegans work with in vitro single-molecule analysis and structural studies of the motor domain. The structure shows that KLP-12 bends tubulin heterodimers to a level that lies in between the extremes of bending by KIF5B (lattice stabilizer) and KIF2C (lattice destabilizer). This study will be of interest to those in the fields of neuronal development and cytoskeletal dynamics.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

Kinesin superfamily proteins are microtubule-based molecular motors driven by the energy of ATP hydrolysis. Among them, the kinesin-4 family is a unique motor that inhibits microtubule dynamics. Although mutations of kinesin-4 cause several diseases, its molecular mechanism is unclear because of the difficulty of visualizing the high-resolution structure of kinesin-4 working at the microtubule plus-end. Here, we report that KLP-12, a C. elegans kinesin-4 ortholog of KIF21A and KIF21B, is essential for proper length control of C. elegans axons, and its motor domain represses microtubule polymerization in vitro. The crystal structure of the KLP-12 motor domain complexed with tubulin, which represents the high-resolution structural snapshot of the inhibition state of microtubule-end dynamics, revealed the bending effect of KLP-12 for tubulin. Comparison with the KIF5B-tubulin and KIF2C-tubulin complexes, which represent the elongation and shrinking forms of microtubule ends, respectively, showed the curvature of tubulin introduced by KLP-12 is in between them. Taken together, KLP-12 controls the proper length of axons by modulating the curvature of the microtubule ends to inhibit the microtubule dynamics.

Article activity feed

  1. Version published to 10.7554/elife.77877 on eLife
  2. eLife

    Author Response

    Reviewer #2 (Public Review):

    Taguchi et al. carried out a functional and structural analysis of microtubule dynamics inhibition by the C. elegans kinesin-4 KLP-12. The authors found that both the motor domain and the tail of KLP-12 are necessary to precisely control axon length in C. elegans. The authors showed that a minimal dimer of KLP-12 is motile along the microtubule lattice and reduces microtubule growth rate in vitro; further biochemistry assay demonstrated that the KLP-12 motor domain can similarly bind the microtubule lattice and free tubulin. The authors then solved the crystal structure of KLP-12 motor domain in complex with tubulin and compared their structure data with that of Kif5B (a motile kinesin that does not depolymerize microtubules) and Kif2C (not actively motile but depolymerizes microtubules). They found that the structure of KLP-12 is more similar to that of Kif5B than that of Kif2C, whereas the curvature of tubulin in complex with KLP-12 is between the curvatures of tubulin in complex with Kif5B and Kif2C. The high-resolution structural data from this study suggest how kinesin-4 can be motile along the microtubule lattice and at the same time stop the microtubule dynamics at its plus end; the mild effect of KLP-12 on protofilament bending may be crucial in enabling the inhibition of both the polymerization and depolymerization of the microtubules.

    Overall, this is a very nice study, although some aspects of data analysis or interpretation need to be extended or clarified.

    We sincerely appreciate the kind and fair evaluation of this reviewer.

    1. Microtubule dynamics may be inhibited by reducing growth rate, inducing pausing, or altering catastrophe. To make their results more solid, the authors should examine whether KLP-12 impacts microtubule pausing and/or catastrophe. Such additional metrics may help strengthen the results and further the insight into the role of tubulin curvature in microtubule dynamics.

    We thank this reviewer for the constructive suggestion. We evaluated each factor and found growth rate is the most affected but depolymerization rate was not significantly affected. The frequency of MT catastrophe events was slightly reduced (Figure 2G). This is similar to the result of KIF21A- or KIF5-bound microtubules suggesting the property is conserved in a broad range of kinesins. Frequency of rescue events was reduced as well (Fig 2I). One possibility is that KLP-12 suppresses microtubule polymerization. Another possibility is the indirect effect induced by reduced MT catastrophe events. We have included these in the result section (pages 8-9, line 187-204; Figure 2).

    1. Structural comparison may be sensitive to the resolution of protein structures that are compared. The authors solved the crystal structure of KLP-12 at a resolution of 2.9 A, which is different from that of Kif4, Kif5B, or Kif2C from previous structure studies (1.7, 3.2, and 3.2 A). The values of root-mean-square distance between protein structures tend to increase if the two proteins that are being compared have been resolved at different resolutions. To strengthen their structural comparison results, the authors should account for the effect of different crystallographic resolutions on their root-mean-square distance evaluations.

    We agree that the resolution of protein structures is important for the rmsd comparison. Thus, we have re-calculated the rmsd values for a fair comparison using the main chain residues (page 13, lines 310-312; Figure 4A).

    1. Structural comparison may also be sensitive to what the proteins are in complex with. The authors solved the structure of KLP-12 that is in complex with GTP-tubulin, which may be different from the structure of KLP-12 that is free of tubulin, or in complex with GDP-tubulin. Previous studies had solved the structure of Kif4 which is free of tubulin (Chang et al 2013), and the structures of Kif5B (Gigant et al 2013) and Kif2C in the presence of GDP (Wang et al 2017). To strengthen their results, the authors should clarify how these differences between the previous and the current structural studies impact their structural comparison results.

    As this reviewer suggested, the kinesin conformations are affected by the nucleotide state of the motor, by forming a complex with tubulin or microtubule, and the nucleotide state of tubulin or microtubule. Thus, we have compared the KLP-12–GTP-tubulin complex with available kinesin-4 structures, kinesin-1 structures, and kinesin-13 structure. These comparisons are shown in the revised Figure 4 and Figure4–supplement 1, demonstrating what is specific for KLP-12 or what is common among kinesin-4.

  3. eLife

    Evaluation Summary:

    Here, Taguchi et al. study a member of the kinesin-4 family of motors, which is important in controlling microtubule length during normal development and maintenance. The authors aim to determine how a member of the kinesin-4 family is able to stabilize the tips of microtubules to suppress both their growth and shrinkage. This paper provides compelling data on KLP-12 by combining in vivo C. elegans work with in vitro single-molecule analysis and structural studies of the motor domain. The structure shows that KLP-12 bends tubulin heterodimers to a level that lies in between the extremes of bending by KIF5B (lattice stabilizer) and KIF2C (lattice destabilizer). This study will be of interest to those in the fields of neuronal development and cytoskeletal dynamics.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    This asks how members of the kinesin-4 family stabilise the tips of microtubules so as to suppress both their growth and shrinkage. Using crystallography of soluble tubulin in complex with KLP-12, a kinesin-4 from C. elegans, electron microscopy, and biochemistry, together with characterisation of mutants in C. elegans, the authors build a case that kinesin-4 motors control microtubule length by stabilising tubulin in a bent conformation that is too straight to depolymerise readily and too bent to insert stably into the lattice.

    The core of the work is a new crystal structure of KIF12 in complex with GMPCPP tubulin, with assembly of the tubulin blocked with a DARPIN that is fused to the kinesin. The structure is 2.9A resolution. Evidence is also presented that the KIF12 controls microtubule length in neurons and in vitro, that it is motile when artificially dimerised, and that its ATPase is activated both by microtubules and by free tubulin - especially by free tubulin.

    The structure shows that KIF12 bends tubulin heterodimers to a level that lies between the extremes of bending defined by the KIF5B (lattice stabiliser) and KIF2C (lattice destabiliser) complexes. The detailed structure of the KIF12-tubulin interface is compared with these exemplars and KIF12 / kinesin-4 specific features identified.

    The work will impact thinking about the detailed molecular basis of the mechanical interaction of kinesins with tubulins.

  5. eLife

    Reviewer #2 (Public Review):

    Taguchi et al. carried out a functional and structural analysis of microtubule dynamics inhibition by the C. elegans kinesin-4 KLP-12. The authors found that both the motor domain and the tail of KLP-12 are necessary to precisely control axon length in C. elegans. The authors showed that a minimal dimer of KLP-12 is motile along the microtubule lattice and reduces microtubule growth rate in vitro; further biochemistry assay demonstrated that the KLP-12 motor domain can similarly bind the microtubule lattice and free tubulin. The authors then solved the crystal structure of KLP-12 motor domain in complex with tubulin and compared their structure data with that of Kif5B (a motile kinesin that does not depolymerize microtubules) and Kif2C (not actively motile but depolymerizes microtubules). They found that the structure of KLP-12 is more similar to that of Kif5B than that of Kif2C, whereas the curvature of tubulin in complex with KLP-12 is between the curvatures of tubulin in complex with Kif5B and Kif2C. The high-resolution structural data from this study suggest how kinesin-4 can be motile along the microtubule lattice and at the same time stop the microtubule dynamics at its plus end; the mild effect of KLP-12 on protofilament bending may be crucial in enabling the inhibition of both the polymerization and depolymerization of the microtubules.

    Overall, this is a very nice study, although some aspects of data analysis or interpretation need to be extended or clarified.

    1. Microtubule dynamics may be inhibited by reducing growth rate, inducing pausing, or altering catastrophe. To make their results more solid, the authors should examine whether KLP-12 impacts microtubule pausing and/or catastrophe. Such additional metrics may help strengthen the results and further the insight into the role of tubulin curvature in microtubule dynamics.

    2. Structural comparison may be sensitive to the resolution of protein structures that are compared. The authors solved the crystal structure of KLP-12 at a resolution of 2.9 A, which is different from that of Kif4, Kif5B, or Kif2C from previous structure studies (1.7, 3.2, and 3.2 A). The values of root-mean-square distance between protein structures tend to increase if the two proteins that are being compared have been resolved at different resolutions. To strengthen their structural comparison results, the authors should account for the effect of different crystallographic resolutions on their root-mean-square distance evaluations.

    3. Structural comparison may also be sensitive to what the proteins are in complex with. The authors solved the structure of KLP-12 that is in complex with GTP-tubulin, which may be different from the structure of KLP-12 that is free of tubulin, or in complex with GDP-tubulin. Previous studies had solved the structure of Kif4 which is free of tubulin (Chang et al 2013), and the structures of Kif5B (Gigant et al 2013) and Kif2C in the presence of GDP (Wang et al 2017). To strengthen their results, the authors should clarify how these differences between the previous and the current structural studies impact their structural comparison results.

  6. eLife

    Reviewer #3 (Public Review):

    The authors study kinesin-4 KLP-12 using in vivo mutants and reconstitution of microtubule dynamics in vitro and show that KLP-12 prevents microtubule polymerisation. The authors then explain these observations with a crystal structure of KLP-12 motor domain in complex with a tubulin dimer. Using this structure and by comparing it to kinesin-1 KIF5B which binds straight tubulin dimers and kinesin-13 KIF2C which depolymerizes microtubules, they show how KLP-12 bends the tubulin dimer and explain how this bending mechanism can prevent polymerization.

    Strengths:

    The crystal structure of the motor domain in complex with a tubulin dimer adds a precious new structure of a motor bound to a tubulin dimer in a new conformation. The comparison with the structures of kinesin-1 KIF5B and kinesin-13 KIF2C also bound to a tubulin dimer is very thorough and convincing. The conserved residues mediating the interaction with tubulin are highlighted. This study adds a new type of curvature induced by a motor protein that influences microtubule dynamics. This work adds to the knowledge of the regulation of microtubule dynamics by microtubule motors.

    Weaknesses:

    Overall, the results support the conclusions. However, more analyses could be done to strengthen the authors' claims.
    The authors start the study using mutants of full-length KLP-12 that they study in vivo in C. elegans. It is however unclear which mutations were made and what their predicted effects on the structure/function of the protein are.
    In Figure 2, the dynamic microtubule assays show that a truncated construct of KLP-12 reduces the microtubule growth rate. However, the authors do not show whether KLP-12 has an effect on other parameters of microtubule dynamics thereby limiting the authors' claims.

  7. Version published to 10.1101/2022.02.14.480441v1 on bioRxiv