Kinesin-1 conformational dynamics are controlled by a cargo-sensitive TPR switch
- School of Biochemistry and Biomedical Sciences, University of Bristol, Biomedical Sciences Building, University Walk, United Kingdom
- School of Chemistry, University of Bristol, Cantock’s Close, United Kingdom
- School of Engineering Mathematics and Technology, University of Bristol, United Kingdom
- Living Systems Institute, University of Exeter, Stocker Road, United Kingdom
- Department of Biosciences, University of Exeter, Stocker Road, United Kingdom
- GW4 Facility for High-Resolution Electron Cryo-Microscopy, University of Bristol, United Kingdom
- Max Planck-Bristol Centre for Minimal Biology, University of Bristol, Cantock’s Close, United Kingdom
Figures
Figure 1 with 4 supplements
Computational modelling and single particle electron microscopy (EM) analysis identify a tetratricopeptide repeat (TPR) docking site (TDS) on CC1.
(A) Schematic representation of heterotetramer kinesin-1 in an open conformation. (B) AF3 model in three orientations alongside a schematic showing kinesin heavy chain (KHC) (KIF5C) coiled-coil assembly. KHCs are shown in blue and cyan, KLCs in orange and purple. The complete AF3 output, including models for the full tetramer and sequences used are described in Figure S1. The schematic shows predicted domain positioning, omitting linker sequences and separating the KHC coiled-coils for clarity (C) Representative NS electron micrograph (from three independent experiments) showing ElbowLock complexes (scale bar is 50 nm) with selected 2D classes from negative stain electron microscopy (NS-EM) (scale bar is 26 nm). (D) Reference-free 2D class averages showing two orientations of complete heterotetrameric kinesin-1 complexes, compared to low-pass filtered back-projection of the AF3 model (scale bar is 22 nm). (E) Structure of the nanobody-TPR complex highlighting binding on exterior of TPRs 4 and 5 (pdb:6fuz). Cargo JIP1 Y-acidic peptide was removed for clarity. (F) Representative NS electron micrograph showing ElbowLock-nanobody complexes (scale bar is 50 nm) with selected 2D classes from NS-EM (scale bar is 26 nm).
Figure 1—figure supplement 1
AlphaFold3 models of KHC-KLC complexes.
(A) Models of kinesin-1 heterotetramer coiled-coil domains (KIF5C CC1-CC4; aa 410–917) with two kinesin light chains (KLCs) (KLC1A CC-TPR; aa 1–480). (B) Models of complete full-length (KIF5C/KLC1A) kinesin-1 heterotetramers, including motor domains and C-terminal KLC sequences. (C) Representative predicted aligned error plot highlighting confidence in intra and inter-domain interactions in a full-length heterotetrameric model. (D) Representative model showing pLDDT. Plots and confidence statistics are typical of other models.
Figure 1—figure supplement 2
Representative samples of protein complexes used in this study.
Coomassie-stained SDS-PAGE gels show samples of proteins after size-exclusion chromatography, with and without BS3 crosslinker (top panels). Coomassie-stained SDS-PAGE gel showing purification of ElbowLock-Nanobody complex from size-exclusion chromatography (bottom panel).
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Figure 1—figure supplement 2—source data 1
Labelled full gel images relating to Figure 1—figure supplement 2.
- https://cdn.elifesciences.org/articles/109462/elife-109462-fig1-figsupp2-data1-v1.pdf
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Figure 1—figure supplement 2—source data 2
Raw files for Coomassie gel images relating to Figure 1—figure supplement 2.
- https://cdn.elifesciences.org/articles/109462/elife-109462-fig1-figsupp2-data2-v1.zip
Figure 1—figure supplement 3
Negative stain electron microscopy (NS-EM) workflow.
Schematic shows typical workflow for 2D classification steps to process NS-EM. Example shown is for ElbowLock complexes.
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Figure 1—figure supplement 3—source data 1
Labelled full gel images relating to Figure 1—figure supplement 2.
- https://cdn.elifesciences.org/articles/109462/elife-109462-fig1-figsupp3-data1-v1.pdf
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Figure 1—figure supplement 3—source data 2
Raw files for Coomassie gel image relating to Figure 1—figure supplement 2.
- https://cdn.elifesciences.org/articles/109462/elife-109462-fig1-figsupp3-data2-v1.zip
Figure 1—figure supplement 4
Representative sets of 2D classes for all complexes.
The scale bar is 26 nm.
Figure 2 with 2 supplements
The CC1 TPR docking site (TDS) is required for the formation of the kinesin-1 shoulder.
(A) Schematic showing strategy to delete the TDS in CC1 and replace it with an equivalent length of a de novo designed homodimeric coiled coil, CC-Di. (B) Size-exclusion chromatography (SEC) traces showing the elution profile of wild-type, ElbowLock, and ElbowLock-ΔTDS proteins. Constructs in the ElbowLock background elute exclusively in the peak associated with closed lambda particles. Representative of three independent experiments. (C) Representative negative stain (NS) electron micrographs (from three independent experiments) showing ElbowLock and ElbowLock-ΔTDS complexes. Scale bar is 50 nm (D) Selected 2D classes from NS-EM highlighting the presence of the shoulder in ElbowLock but not ElbowLock-ΔTDS complexes. The scale bar is 26 nm. Full sets of classes are provided in Figure 1—figure supplement 4 (E) Quantification of number of particles in classes with and without a prominent shoulder of three independent experiments. *** indicates p<0.001 (F) Fluorescence polarisation binding assays show fluorescently labelled TDS and SKIP (W-acidic motif, positive control) peptides, but not the control peptide CC-Di, bind to isolated TPR domains. Error bars show S.E.M. from three replicates.
Figure 2—figure supplement 1
Alphafold3 models of ΔTDS kinesin-1 complexes.
(A) Five models showing ΔTDS complexes (CCDi replaces the TPR docking site (TDS) sequence shown in green). In all models, the TPR domains are displaced from their original CC1-docked position (compare with Figure 1—figure supplement 1) and occupy a variety of different positions with very low confidence (B) Representative position error plot (from model 1). Box highlights low confidence in TPR positioning.
Figure 2—figure supplement 2
Biophysical characterisation of Eng-TPR docking site (TDS).
(A) Schematic showing design of Eng-TDS construct with flanking CCDi sequence and leucine mutations in the hydrophobic core. (B) Circular dichroism (CD) analysis of Eng-TDS indicates alpha helical structure. (C) Analytical ultracentrifugation (AUC) analysis of Eng-TDS indicates assembly of a dimeric coiled-coil. (D, E) Chromatogram showing the purification of Eng-TDS, accompanied by mass spectrometry analysis confirming a molecular mass of 4895 Da. (F) Fluorescence polarisation (FP) assay showing binding of fluorescently labelled peptides from Eng-TDS and SKIP W-acidic motif to KLC2. (G) FP assay showing no detectable binding of Eng-TDS to KLC2 lacking the first helix of TPR1. (H) Fluorescence polarisation binding assays show no detectable binding of fluorescently labelled TDS and SKIP (W-acidic motif) peptides to isolated KLC1-TPR-KinTag fusion proteins.
Figure 3 with 2 supplements
Adaptor binding to KLC-tetratricopeptide repeat (TPR) dislocates the kinesin-1 shoulder.
(A) Comparison of X-ray crystal structures of ligand-free (grey, pdb: 3nf1) and KinTag (W/Y acidic)-ligand bound (purple, pdb: 6swu) TPR domains highlighting the ligand-binding site and ligand-induced change in TPR curvature. (B) Size-exclusion chromatography (SEC) traces showing elution profile of wild-type, ElbowLock, and ElbowLock-KinTag proteins. (C) Representative NS electron micrographs (from three independent experiments) showing ElbowLock and ElbowLock-KinTag complexes. Scale bar is 50 nm. (D) 2D classes from NS-EM highlighting the loss of the shoulder in ElbowLock-KinTag complexes. The scale bar is 26 nm. (E) Quantification of the percentage of particles in classes with and without a prominent shoulder from three independent experiments. ** indicates p<0.001 and *** indicates p<0.001 (F) 3D reconstructions from the ElbowLock, ElbowLock-ΔTDS, and ElbowLock-KinTag datasets show dislocation of the shoulder either upon deletion of the TDS or incorporation of KinTag. (G) Model showing dislocation of the shoulder induced upon KinTag binding.
Figure 3—figure supplement 1
Representative negative stain electron microscopy (NS-EM) electron micrograph showing wild-type KinTag particles.
Selected open and closed particles are expanded on the right. The scale bar is 50 nm.
Figure 3—figure supplement 2
Electron microscopy (EM) processing workflow for 3D reconstructions.
Figure 4 with 4 supplements
Adaptor binding to the kinesin light chain (KLC) tetratricopeptide repeat (TPR) domain promotes motor accessibility.
Summaries of comparative hydrogen/deuterium-exchange mass spectrometry (HDX-MS) analysis of wild-type, DeltaElbow, ElbowLock, and ElbowLock-Kintag complexes. At each time point, the rates of exchange were subtracted as indicated (X minus Y) and filtered using a hybrid significance test global significance and Welch’s t-test, then transposed onto X-ray crystal structures of the motor domains (pdb: 3kin) and TPR domain (pdb: 3nf1). Grey indicates non-significant differences or limited peptide coverage. (A) Comparison of deltaElbow (X) against wild-type (Y). (B) ElbowLock (X) against wild-type (Y). (C) ElbowLock-Kintag (X) against ElbowLock (Y). Relative protection (blue) indicates less solvent exposure or increased H-bonding for X, while deprotection (red) indicates more solvent exposure or H-bond loss for X.
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Figure 4—source data 1
Hydrogen/deuterium-exchange mass spectrometry (HDX-MS) summary table.
- https://cdn.elifesciences.org/articles/109462/elife-109462-fig4-data1-v1.docx
Figure 4—figure supplement 1
Schematic representation of the hydrogen/deuterium-exchange (HDX) experiment.
Figure 4—figure supplement 2
Coverage map of mass spectral assignment.
Coverage of peptides in ‘bottom-up’ hydrogen/deuterium-exchange mass spectrometry (HDX-MS) experiments for kinesin-1 HC (A) and LC (B).
Figure 4—figure supplement 3
Difference plots showing total hydrogen/deuterium-exchange (HDX) across timepoints comparing heavy chain of DeltaElbow and Elbowlock to wild-type and ElbowLock-KinTag to ElbowLock.
The difference in the total observed deuterium uptake at 0.3, 0.5, 1, 3, 10, 30, and 300 s was summed and compared between DeltaElbow and wild-type (A) and ElbowLock and wild-type (B), and ElbowLock-KinTag to ElbowLock (C). In each case, the data for the later were subtracted from the former. Therefore, relative protection in a region of the former results in a more negative value (blue bars extending downward), while deprotection, such as from an exposed domain interface, results in a more positive value (red bars extending upward). Each vertical bar represents a distinct peptide, and the horizontal axis corresponds to amino acid number from N to C. Significant differences in deuterium labelling per peptide and time point, derived from a T-test, are marked with pink asterisks.
Figure 4—figure supplement 4
Difference plots showing total hydrogen/deuterium-exchange (HDX) across all timepoints comparing light chain of DeltaElbow and Elbowlock to wild-type and ElbowLock-KinTag to ElbowLock.
The difference in the total observed deuterium uptake at 0.3, 0.5, 1, 3, 10, 30, and 300 s was summed and compared between DeltaElbow and wild-type (A) and ElbowLock and wild-type (B), and ElbowLock-KinTag to ElbowLock (C). In each case, the data for the later were subtracted from the former. Therefore, relative protection in a region of the former results in a more negative value (blue bars extending downward), while deprotection, such as from an exposed domain interface, results in a more positive value (red bars extending upward). Each vertical bar represents a distinct peptide, and the horizontal axis corresponds to amino acid number from N to C. Significant differences in deuterium labelling per peptide and time point, derived from a T-test, are marked with pink asterisks.
Figure 5
Opening the kinesin-1 complex promotes its association with MAP7.
(A) Overlay of two Alphafold3 models of a tetrameric CC1/TPR assembly and a tetrameric CC1/MAP7-kinesin binding domain assembly, aligned on CC1. For clarity, only one CC1 model is shown. (B) GFP-MAP7 immunoprecipitation experiments showing enhanced association of kinesin heavy chain (KHC) and kinesin light chain (KLC) in the DeltaElbow background. Cells were transfected with the indicated constructs GFP/GFP-MAP7, HA-KLC2, HA-KIF5C (WT, ElbowLock, DeltaElbow). Complexes were immunoprecipitated using GFP-TRAP beads, and input and bound samples were analysed using western blotting with anti-GFP and anti-HA antibodies. (C) Quantification of b. from 4 independent experiments. Error bars show S.E.M. ***p<0.001 using one-way ANOVA with Tukey’s multiple comparison test. (D) Immunofluorescence images of HeLa cells transfected with GFP-MAP7, HA-KHC, and KLC2-Halo (TMR-labelled) showing enhanced association of kinesin-1 with GFP-MAP7 positive microtubules in the DeltaElbow background. Images are representative of three independent experiments. Scale bar is 20 µm.
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Figure 5—source data 1
Uncropped western blots relating to Figure 5B.
- https://cdn.elifesciences.org/articles/109462/elife-109462-fig5-data1-v1.pdf
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Figure 5—source data 2
Raw TIFF files for western blots relating to Figure 5B.
- https://cdn.elifesciences.org/articles/109462/elife-109462-fig5-data2-v1.zip
Figure 6
Model for cargo-mediated initiation of kinesin-1 activation.
In the autoinhibited state, a tetratricopeptide repeat (TPR) domain(s) are docked at the TPR docking site (TDS) forming the shoulder. (i). Binding to the cargo adaptor dislocates the TPR shoulder and promotes motor domain accessibility, and initiates separation of the coiled-coil domains (ii). This allosteric coupling between cargo and motor would facilitate recruitment to microtubules (supported by MAP7) and subsequent transport (iii).
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Recombinant DNA reagent | pMW-His-KIF5C | Weijman et al., 2022 | pMW vector, N-terminal 6×His tag, for bacterial expression. | |
| Recombinant DNA reagent | pET28-KLC1 | Weijman et al., 2022 | pET28, His tag removed, for bacterial expression. | |
| Recombinant DNA reagent | pMW-His-KIF5C (ElbowLock) | Cross et al., 2024 | KIF5C, elbow modification, for bacterial expression. | |
| Recombinant DNA reagent | pMW-His-KIF5C (DeltaElbow) | Weijman et al., 2022 | KIF5C, elbow modification, for bacterial expression. | |
| Recombinant DNA reagent | pMW-His-KIF5C-ΔTDS | This paper | KIF5C, CC1 modification, for bacterial expression. | |
| Recombinant DNA reagent | KLC1-KinTag | This paper | pET28, His tag removed, for bacterial expression, C-terminal KinTag fusion via (TGS)₁₀ linker | |
| Recombinant DNA reagent | KLC1 TPR domain | Pernigo et al., 2013 | pET28, His tagged KLC1 TPR domain | |
| Recombinant DNA reagent | KLC2 TPR domain | Pernigo et al., 2013 | pET28, His tagged KLC2 TPR domain | |
| Recombinant DNA reagent | KLC2 TPR domain delta Helix 1 | Pernigo et al., 2013 | pET28, His tagged KLC2 TPR domain, helix 1 removed | |
| Recombinant DNA reagent | Anti-TPR nanobody | Pernigo et al., 2018 | Synthesised (GenScript), pelB leader, C-terminal His tag | |
| Recombinant DNA reagent | GFP-MAP7 | Metzger et al., 2012 | Addgene plasmid #46076 | Mammalian expression |
| Recombinant DNA reagent | HA-KLC2 | Sanger et al., 2017 | Mammalian expression | |
| Recombinant DNA reagent | HA-KIF5C | Sanger et al., 2017 | Mammalian expression | |
| Recombinant DNA reagent | KLC2-Halo | Yip et al., 2016 | Mammalian expression | |
| Recombinant DNA reagent | HA-KIF5C DeltaElbow | Weijman et al., 2022 | Mammalian expression | |
| Recombinant DNA reagent | HA-KIF5C ElbowLock | Cross et al., 2024 | Mammalian expression |
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Kinesin-1 conformational dynamics are controlled by a cargo-sensitive TPR switch
eLife 14:RP109462.
https://doi.org/10.7554/eLife.109462.3
