Figures and data
Computational modelling and single particle EM analysis identify a TPR docking site (TDS) on CC1
(A) Schematic representation of heterotetramer kinesin-1 in an open conformation. (B) AF3 model in 3 orientations alongside a schematic showing KHC(KIF5C) coiled-coil assembly with KLCs. KHCs are shown in blue and cyan, KLCs in orange and purple. The complete AF3 output and sequences used are described in Figure S1. Schematic shows predicted domain positioning, omitting linker sequences and separating the KHC coiled-coils for clarity (C) Representative NS electron micrograph (from 3 independent experiments) showing ElbowLock complexes (scale bar is 50 nm) with selected 2D classes from NS-EM (scale bar is 22 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 22 nm).
The CC1 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) 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 3 independent experiments. (C) Representative NS electron micrographs (from 3 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 22 nm. Full sets of classes are provided in Fig. S4 (E) Quantification of number of particles in classes with and without a prominent shoulder of 3 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 3 replicates.
Adaptor-binding to KLC-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) SEC traces showing elution profile of wild type, ElbowLock and ElbowLock-KinTag proteins. (C) Representative NS electron micrographs (from 3 independent experiments) showing ElbowLock and ElbowLock-KinTag complexes. Scale bar = 50 nm. (D) 2D classes from NS-EM highlighting the loss of the shoulder in ElbowLock-KinTag complexes. (E) Quantification of the percentage of particles in classes with and without a prominent shoulder from 3 independent experiments. ** indicates p 0.001 and *** indicates p < 0.001(E) 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. (F) Model showing dislocation of the shoulder induced upon KinTag binding.
Adaptor-binding to the KLC TPR domain promotes motor accessibility.
Summaries of comparative 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 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.
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 KHC and KLC in the DeltaElbow background. (C) Quantification of b. from 4 independent experiments. Error bars show S.E.M. ***=p<0.001 using 1-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 enhance association of kinesin-1 with GFP-MAP7 positive microtubules in the DeltaElbow background. Images are representative of 3 independent experiments. Scale bar is 20 µm (e) Model: In the autoinhibited state, a TPR domain(s) are docked at the TDS forming the shoulder i. Binding to the cargo adaptor dislocates the TPR shoulder and promotes motor domain accessibly, 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).
Alphafold3 models of KHC-KLC complexes.
(A) Models of kinesin-1 heterotetramer coiled-coil domains (KIF5C CC1-CC4; aa 410-917) with two KLCs (KLC1A CC-TPR; aa 1-480). (C) 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. Plot is typical of other models.
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 panel). Coomassie-stained SDS-PAGE gel showing purification of ElbowLock-Nanobody complex from size-exclusion chromatography (bottom panel).
Representative NS-EM processing workflow.
Schematic shows typical workflow for 2D classification steps to process NS-EM. Example shown is for ElbowLock complexes.
Eng-TDS design, characterisation and interaction with KLC.
(A) Schematic showing design of Eng-TDS construct with flanking CCDi sequence and leucine mutations in the hydrophobic core. (B) CD analysis of Eng-TDS indicates alpha helical structure. (C) 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) 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.
Representative NS-EM electron micrograph showing wild type-KinTag particles with selected open and close particles expanded on right.
The scale bar is 50 nm.
Schematic representation of the HDX experiment.
Difference plots showing total 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 seconds 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.
Difference plots showing total 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 seconds was summed and compared between DeltaElbow and wild type (A) and ElbowLock and wildtype (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.
