Kinesin-1 holoenzyme assembly coordinates cargo-adaptor recognition with heavy-chain autoinhibition
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eLife Assessment
Nui and colleagues tackle several questions concerning the assembly of the heterotetrameric kinesin-1 complex, examining intermolecular interactions between KHC and KLC and KHC with the cargo adaptor TRAK2. Aspects of the study using computational models and biopysical/biochemical analysis to examine the interaction between KHC and KLC and KHC and TRAK2 are useful, alongside an interesting new model for modulation of TRAK2 binding by KLC. The study will be of great interest to the community, but currently it is incomplete, with some claims partially supported.
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Abstract
Kinesin-1 is a major microtubule-based molecular motor that transports diverse cellular cargoes, yet how assembly of its subunits is coupled to cargo recognition and motor regulation remains poorly understood. Mammalian kinesin-1 functions as a heterotetrameric holoenzyme composed of kinesin heavy chains (KIF5s) and kinesin light chains (KLCs), but the structural principles linking holoenzyme assembly to motor regulation remain unclear. Here, using KIF5C as a model system, we define the molecular mechanisms underlying kinesin-1 holoenzyme assembly and cargo-adaptor regulation. We identify a conserved coiled-coil interface between KIF5C and KLC1 that mediates their 2:2 assembly, with quantitative mutagenesis revealing key hydrophobic determinants of complex formation. We further demonstrate that the mitochondrial adaptor TRAK2 directly engages the KIF5C CC4 cargo-binding platform through a defined 2:2 interaction required for mitochondrial recruitment of KIF5C. Although KLC1 and TRAK2 bind distinct regions of KIF5C, KLC1 modulates TRAK2 association through steric and conformational effects, revealing how holoenzyme composition influences cargo-adaptor accessibility. Mechanistically, we identify a previously unrecognized intramolecular interaction between the KIF5C CC1 and CC4 domains that forms a stalk-mediated autoinhibitory latch. TRAK2 and KLC1 release this inhibitory interaction through distinct mechanisms. Together, our study establishes a molecular framework in which kinesin-1 holoenzyme assembly regulates cargo-adaptor recognition and autoinhibitory remodeling, providing mechanistic insight into how molecular motors coordinate cargo engagement with activation.
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eLife Assessment
Nui and colleagues tackle several questions concerning the assembly of the heterotetrameric kinesin-1 complex, examining intermolecular interactions between KHC and KLC and KHC with the cargo adaptor TRAK2. Aspects of the study using computational models and biopysical/biochemical analysis to examine the interaction between KHC and KLC and KHC and TRAK2 are useful, alongside an interesting new model for modulation of TRAK2 binding by KLC. The study will be of great interest to the community, but currently it is incomplete, with some claims partially supported.
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Reviewer #1 (Public review):
Niu and colleagues tackle several questions concerning the assembly of the heterotetrameric kinesin-1 complex, comprising kinesin heavy chains (KHCs) and kinesin light chains (KLCs), its interaction with adaptor proteins (the mitochondrial adaptor TRAK2), and the implications of these interactions for kinesin autoregulation.
Broadly, the study comprises four parts; strengths and weaknesses are described for each in turn.
First, the authors examine the coiled-coil interface that enables tight binding between KHC and KLC. Binding-site mapping supports previous observations that KHC and KLC interact through the CC3 region of KHC and the amino-terminal coiled-coils of KLC. These regions are modelled using AlphaFold3, and the resulting models are used to inform mutagenesis experiments that identify critical …
Reviewer #1 (Public review):
Niu and colleagues tackle several questions concerning the assembly of the heterotetrameric kinesin-1 complex, comprising kinesin heavy chains (KHCs) and kinesin light chains (KLCs), its interaction with adaptor proteins (the mitochondrial adaptor TRAK2), and the implications of these interactions for kinesin autoregulation.
Broadly, the study comprises four parts; strengths and weaknesses are described for each in turn.
First, the authors examine the coiled-coil interface that enables tight binding between KHC and KLC. Binding-site mapping supports previous observations that KHC and KLC interact through the CC3 region of KHC and the amino-terminal coiled-coils of KLC. These regions are modelled using AlphaFold3, and the resulting models are used to inform mutagenesis experiments that identify critical residues within the hydrophobic core of the interface. Although the binding site has previously been reasonably well defined, both biochemically and using computational models, this work extends those findings to individual residues at the interface and provides supporting biophysical analysis. This provides useful experimental validation of the computational models and important tools for the field to enable the selective uncoupling of KHC and KLC.
Second, the authors examine how the mitochondrial adaptor TRAK2 interacts with KHC. Previous studies have defined the critical regions of KHC CC4 and TRAK2 reasonably well, and the authors' biochemical mapping broadly supports these findings. They extend this through biophysical analysis and computational modelling that proposes a 2:2 stoichiometry and an interesting assembly in which CC4 separates to enable formation of a four-helix bundle with TRAK2. This AlphaFold model is supported by biophysical, biochemical, and cell-imaging analyses, including the identification of point mutations that disrupt the interaction. These findings are important because the molecular basis of KHC CC4 interactions with diverse cargo adaptors has remained somewhat opaque, and this study begins to place these interactions on a firmer molecular footing. Some of the principles proposed here may also extend to other KHC-binding adaptors.
Third, and of particular interest, the authors examine the interplay between KLC and TRAK binding to KHC. This is important because previous work has generally favoured the view that mitochondrial transport is independent of KLCs, although some studies disagree. Whether KLCs are excluded from TRAK-containing complexes and vice versa and, if so, how this occurs has remained unclear. The authors provide evidence suggesting that the extended coiled-coil region of KLC, outside the direct high-affinity KHC-binding interface, modulates CC4 to prevent TRAK binding, suggesting that KHC-TRAK and KHC-KLC complexes may be mutually exclusive. Precisely how this mechanism translates into transport regulation within the cell remains unclear, but the effort to establish a structural hypothesis for understanding this interplay is valuable.
Fourth, the authors provide evidence for a novel, weak interaction between CC1 and CC4 that they propose may contribute to kinesin-1 autoinhibition and its regulation by KLC and TRAK-an 'autoinhibitory latch'. I found this part of the work more preliminary because, although the biophysical and biochemical analyses provide some support for such an interaction, unlike other aspects of the study, it is less well supported by computational modelling or mutational analysis. Moreover, there is no direct evidence for how this interaction might function in the context of full-length kinesin-1 autoinhibition, for example through assays of motor activity or conformational state, and the proposed model is not particularly clear with respect to full-length KLC (including its TPR domains) or regulation by MAP7.
Taken together, the study makes a valuable contribution to our understanding of several facets of kinesin-1 assembly, cargo recognition, and regulation. Key strengths lie in the complementary biochemical, biophysical, computational, and cell-imaging approaches used to support most of the key findings, together with the definition of several very useful point mutants that should inform future work.
There are, however, some general weaknesses surrounding unnecessary or overstated claims of novelty and the connection to prior work, and some factual errors. These can be straightforwardly addressed through better integration of the existing literature into the narrative, without detracting from the authors' contributions. Some conclusions appear to extend beyond the available data, and these should be tempered accordingly. Computational models should also be presented more thoroughly and systematically, together with confidence metrics and multiple ranked outputs. The Methods should more clearly describe the constructs and sequences used so that the work can be straightforwardly reproduced, alongside information on replication and additional quantification. Some clarification around measured stoichiometries and the authors' interpretation for some of the short coiled-coil fragments will also be important.
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Reviewer #2 (Public review):
Summary:
Niu et al. develop a biochemical pipeline using systematic co-immunoprecipitation from cell lysate coupled with in vitro binding studies to test hypotheses generated based on structure prediction of how the kinesin heavy chain is autoinhibited through intramolecular interactions, as well as interactions with the kinesin light chain. Additionally, the authors provide insight into kinesin heavy chain binding to the cargo adaptor TRAK. Structural understanding of kinesin regulation has been elusive due to apparent flexibility of the motor. The advent of protein structure prediction, validated through cross-linking mass spectrometry (XL-MS), has provided an inroad to understanding kinesin regulation (Tan et al., Weijman et al.). Most recently, Ashaduzzaman et al. have used cross-linking and local …
Reviewer #2 (Public review):
Summary:
Niu et al. develop a biochemical pipeline using systematic co-immunoprecipitation from cell lysate coupled with in vitro binding studies to test hypotheses generated based on structure prediction of how the kinesin heavy chain is autoinhibited through intramolecular interactions, as well as interactions with the kinesin light chain. Additionally, the authors provide insight into kinesin heavy chain binding to the cargo adaptor TRAK. Structural understanding of kinesin regulation has been elusive due to apparent flexibility of the motor. The advent of protein structure prediction, validated through cross-linking mass spectrometry (XL-MS), has provided an inroad to understanding kinesin regulation (Tan et al., Weijman et al.). Most recently, Ashaduzzaman et al. have used cross-linking and local refinement to obtain a reconstruction of the autoinhibited KHC:KLC tetramer. The biochemical analysis presented here extends previous studies and has enabled the authors to define the hierarchy of competing KHC interactions with KLC and the TRAK2 adaptor.
Strengths:
While other studies relied predominantly on XL-MS to validate autoinhibitory contacts in the KHC:KLC autoinhibited tetramer, the present study used in-cell interactions to define minimal interacting units, and measured the effects of prediction-guided mutagenesis on their interaction in vitro. This complementary approach stratifies the multitude of predicted autoinhibitory interactions and offers a functional test for reported structures and structure predictions.
Weaknesses:
The work is not appropriately placed in the context of previous literature. The KHC:KLC assembly interface (Figures 1 and 2) was previously predicted by Weijman et al., supported by XL-MS in Tan et al., and subsequently visualized by cryo-EM in Ashaduzzaman et al. Contacts between the regions designated in this study as KHC CC1 and CC4 (Figure 6) were also reported by Tan et al. and Ashaduzzaman et al. Similarly, a structural model of TRAK2:KHC (Figures 3-4) was previously reported and tested by co-immunoprecipitation and mutagenesis by Gladkova et al. Tan et al. additionally mapped binding of the related adaptor TRAK1 to KIF5B using XL-MS. These results should be discussed throughout to help the reader understand whether biochemical observations are consistent with prior work, or whether they hint at previously unknown regulatory mechanisms.
To test the KHC:KLC and KHC:TRAK2 predicted interfaces, mutations of hydrophobic residues were introduced in KHC (Figures 2 and 4), and TRAK2 (Figure 4). However, both KHC and TRAK2 exist as homodimers in solution. Are these mutations specific to destabilizing the heterotetramers, or do they also affect the KHC and TRAK2 homodimers? Such an effect could provide an alternative explanation for reduced binding and impaired mitochondrial recruitment of KIF5C by TRAK2 mutants, such as L217Q/L220Q. Retained mitochondrial localization of TRAK2 mediated by the C-terminus is not a good proxy for functionality of the N-terminal motor-binding region. Mutants should be assessed for stability and dimerization propensity, and their effects fully described.
Several structure-prediction models are introduced without pLDDT and PAE confidence plots. These are necessary to assess structure prediction confidence. Additionally, residue number labels would help orient the reader as to the boundaries of the protein fragments modelled.
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Reviewer #3 (Public review):
The authors report an interaction between KHC and KLC1 through the coiled coil 3 of KHC, and then characterise the binding of TRAK2 to KHC through coiled coil 4 of KHC. The reporting of this second interaction is novel. The authors make a number of mutants which change the affinity of TRAK2 for KHC, but it is not clear if it ultimately prevents binding. They then demonstrate through competitive binding that TRAK2 binds more tightly to CC4 than CC1 and therefore can trigger release of Kinesin-1 auto-inhibition. The authors, however, do not show directly that the kinesin is no longer inhibited. This could be tested using negative stain EM, SAXS or motility assays in the future. The experiments are performed with immunoprecipitation pulldowns coupled with western blotting in cells, and ITC with recombinantly …
Reviewer #3 (Public review):
The authors report an interaction between KHC and KLC1 through the coiled coil 3 of KHC, and then characterise the binding of TRAK2 to KHC through coiled coil 4 of KHC. The reporting of this second interaction is novel. The authors make a number of mutants which change the affinity of TRAK2 for KHC, but it is not clear if it ultimately prevents binding. They then demonstrate through competitive binding that TRAK2 binds more tightly to CC4 than CC1 and therefore can trigger release of Kinesin-1 auto-inhibition. The authors, however, do not show directly that the kinesin is no longer inhibited. This could be tested using negative stain EM, SAXS or motility assays in the future. The experiments are performed with immunoprecipitation pulldowns coupled with western blotting in cells, and ITC with recombinantly purified proteins.
Strengths:
The data showing the binding of KLC1 to KHC and the TRAK2 binding to KHC are interesting.
The ITC data looks very good from the profiles. It seems, however, that the experiments were done once rather than replicated (at least twice, ideally three times to accommodate variation). The size-exclusion chromatography experiments showing competition between different adaptors to KHC are nicely done, and the SEC-MALS is informative in terms of determining the stoichiometry and size of the assembled complexes. The work is performed with recombinant proteins and therefore removes any ambiguity about the interactions in cells.
Weaknesses:
The experiments lack appropriate controls, with some experiments incomplete, and the statements claimed are not supported by the data. There are some problems with figure and data presentation and the number of replicates/lack of quantification. The validation of AlphaFold models and predictions is missing. The cell biology experiments are preliminary.
There is a concern with the cell biology experiments. All experiments are done with transfections in the presence of the endogenous proteins. It is not clear if the proteins are overexpressed and they appear cytoplasmic. Beyond this, it is not possible to claim co-localization of the transfected proteins.
The manuscript would benefit from general editing to improve understanding. The names used to describe the methods are incorrect. This has implications for the solidity of the statements and conclusions of the paper.
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Author response:
We thank the editors and reviewers for their careful and constructive evaluation of our manuscript. We are encouraged that the reviewers recognize the value of our biochemical, biophysical, structural-modeling, and cell-based analyses of kinesin-1 holoenzyme assembly, KHC–TRAK2 recognition, and the regulatory interplay between KLC1 and TRAK2. We also appreciate the concerns regarding the relationship of our findings to previous work, the strength of several mechanistic conclusions, validation and presentation of the structural models, and the need for additional controls and quantitative analyses.
We plan to revise the manuscript substantially in response to these comments. Our provisional responses and planned revisions are summarized below.
Reviewer #1
We agree that the novelty of several findings should be more …
Author response:
We thank the editors and reviewers for their careful and constructive evaluation of our manuscript. We are encouraged that the reviewers recognize the value of our biochemical, biophysical, structural-modeling, and cell-based analyses of kinesin-1 holoenzyme assembly, KHC–TRAK2 recognition, and the regulatory interplay between KLC1 and TRAK2. We also appreciate the concerns regarding the relationship of our findings to previous work, the strength of several mechanistic conclusions, validation and presentation of the structural models, and the need for additional controls and quantitative analyses.
We plan to revise the manuscript substantially in response to these comments. Our provisional responses and planned revisions are summarized below.
Reviewer #1
We agree that the novelty of several findings should be more carefully defined in the context of the existing literature. In the revised manuscript, we will better integrate previous biochemical and structural studies of KHC–KLC assembly, TRAK-family adaptor binding to KHC, and kinesin-1 autoinhibition. In particular, we will explicitly compare our findings with the work of Diefenbach et al. [1]., Weijman et al. [2]., Tan et al. [3]., Ashaduzzaman et al. [4]., and Gladkova et al. [5]., and distinguish previously established structural features from the additional residue-level mutational validation and quantitative biophysical analyses provided in our study. We will accordingly revise statements of novelty throughout the manuscript.
We also agree that our current data establish a direct CC1–CC4 interaction and its disruption by TRAK2 and KLC1, but do not directly demonstrate that this interaction is itself autoinhibitory or that its disruption activates full-length kinesin-1. We will therefore distinguish the experimentally observed CC1–CC4 interaction from the proposed regulatory model and temper terms such as “autoinhibitory latch,” “activation switch,” and related mechanistic conclusions where appropriate. We will also expand the Discussion to consider how this interaction may relate to the established roles of full-length KLC and MAP7 in kinesin-1 regulation.
To strengthen the presentation of the AlphaFold3-based models, we will provide model-confidence information, including pLDDT, PAE, and ipTM metrics, specify the exact sequences and residue boundaries used for each prediction, and include additional prediction outputs where informative. We will also more clearly distinguish experimentally supported conclusions from structural hypotheses, particularly regarding the proposed steric and/or conformational effects of the extended KLC1 coiled-coil on the KIF5C CC4 region.
Finally, we will strengthen the quantitative and methodological presentation of the study. We will provide clearer construct information, explicitly report experimental replication, quantify the mitochondrial enrichment and cellular distribution analyses in Figures 4 and 5, and revisit the interpretation of stoichiometries obtained with shorter coiled-coil fragments. Statements implying mitochondrial transport will be revised to reflect that the current cellular experiments directly demonstrate mitochondrial recruitment rather than organelle motility.
Reviewer #2
We agree that the relationship between our study and previous structural work should be made substantially clearer. Although several relevant studies were cited in the original manuscript, the extent to which our findings confirm, extend, or differ from these previous models was not sufficiently explained. We will revise the Introduction, Results, and Discussion accordingly, including explicit comparisons with the structural predictions of Weijman et al. [2]., the XL-MS-supported model of Tan et al. [3]., the recent cryo-EM/integrative model of Ashaduzzaman et al. [4]., and the TRAK–KHC work of Gladkova et al [5].
We also appreciate the concern that mutations introduced into hydrophobic interfaces could affect the intrinsic stability or homodimerization of KHC or TRAK2, rather than selectively disrupting heterotypic interactions. To address this alternative explanation, we plan to evaluate the oligomeric states and stability of key interface mutants using appropriate biophysical approaches, including SEC-MALS and thermal-stability measurements. These results will be incorporated into the revised manuscript and used to reassess the interpretation of the corresponding binding and cellular recruitment experiments.
As noted above, we will also provide substantially more information on the confidence and reproducibility of the AlphaFold3 predictions, including pLDDT, PAE, and ipTM metrics, and will clearly annotate the residue boundaries of all modeled fragments.
Reviewer #3
We agree that the cellular experiments require additional quantification and clearer presentation. We will quantify the mitochondrial enrichment of KIF5C and the effects of KLC1 co-expression on the intracellular distribution of KIF5C and TRAK2 across independent experiments. We will also include appropriate controls for endogenous/background fluorescence, improve image and scale-bar presentation, and revise the corresponding terminology so that the conclusions accurately reflect the experiments performed.
We will strengthen the reproducibility and presentation of the biochemical and biophysical data. Key ITC measurements will be supported by independent repeats and reported with the corresponding binding parameters and variability. For the SEC experiments in Figure 3, we will include SDS-PAGE analysis of relevant fractions and more clearly describe the composition of each protein mixture. Appropriate positive controls will also be added to the Co-IP experiments where requested.
We will further improve the presentation of the structural models by providing confidence metrics and residue annotations, and we will carefully revise the terminology used to describe SEC, SEC-MALS, ITC, and other biochemical approaches. The Abstract and main text will also be edited to more clearly describe the experimental approaches used and to ensure that all mechanistic conclusions are matched to the strength of the available evidence. More broadly, we will revise the manuscript to sharpen its central message while avoiding overstatement. In particular, we will distinguish direct experimental observations—such as KHC–KLC1 assembly, KHC–TRAK2 recognition, KLC1-dependent modulation of TRAK2 engagement, and the CC1–CC4 interaction—from mechanistic models that remain to be tested more directly.
We expect to complete these revisions and associated experiments within approximately 10–12 weeks. We thank the editors and reviewers again for their constructive comments, which we believe will substantially improve the rigor, clarity, and positioning of the study.
(1) Diefenbach RJ, Mackay JP, Armati PJ, Cunningham AL. The C-terminal region of the stalk domain of ubiquitous human kinesin heavy chain contains the binding site for kinesin light chain. Biochemistry. 1998 Nov 24;37(47):16663-70. doi: 10.1021/bi981163r.
(2) Weijman JF, Yadav SKN, Surridge KJ, Cross JA, Borucu U, Mantell J, Woolfson DN, Schaffitzel C, Dodding MP. Molecular architecture of the autoinhibited kinesin-1 lambda particle. Sci Adv. 2022 Sep 16;8(37):eabp9660. doi: 10.1126/sciadv.abp9660.
(3) Tan Z, Yue Y, Leprevost F, Haynes S, Basrur V, Nesvizhskii AI, Verhey KJ, Cianfrocco MA. Autoinhibited kinesin-1 adopts a hierarchical folding pattern. Elife. 2023 Nov 1;12:RP86776. doi: 10.7554/eLife.86776.
(4) Ashaduzzaman M, Tang Y, Okada K, Fried SD, Mckenney RJ, Al-Bassam J. Structural basis of kinesin-1 autoinhibition and its control of microtubule-based motility. Sci Adv. 2026 Jul 17;12(29):eaeg1267. doi: 10.1126/sciadv.aeg1267.
(5) Gladkova C, Paez-Segala MG, Grant WP, Kittisopikul M, Myers SA, Wang Y, Vale RD. A molecular switch for coordinating kinesin and dynein transport of mitochondrial cargo. Science. 2026 Aug 6;393(6811):601-606. doi: 10.1126/science.aeh1475.
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