Figures and data
KIF5B adopts a hierarchical folding pattern.
(A) Overview of KIF5B domain diagram and the previous model of kinesin-1 autoinhibition. (B) Size exclusion chromatography profile of KIF5B. (C) Negative staining EM analysis of KIF5B in two conformations. (D) Class averages of KIF5B in compact state. (E) End-to-end measurements of KIF5B in two states (N=100). (F) Crosslinked lysine pairs in KIF5B were mapped onto the domain diagram and divided into four groups.
Integrative modeling reveals molecular architecture of autoinhibited KIF5B.
(A) The proposed model of autoinhibited KIF5B in cartoon diagram. The dashed lines indicates the crosslinked pairs. (B) A model of autoinhibited KIF5B via the integrative modeling. The gray density is the 3D reconstruction from the negative staining EM. Three eye icons showed the viewing directions in (C). (C) Three groups of crosslinked pairs were mapped onto the KIF5B model.
KLC1 makes extensive contacts with KIF5C within the inhibited state.
(A) Overview of KIF5C-KLC1 domain diagram. (B) Size exclusion chromatography profile of KIF5C-KLC1. (C) Negative staining EM analysis of Kif5C-KLC1 in two states. (D) Class averages of KIF5C-KLC1 in compact conformation. (E) End to end length measurements of KIF5C-KLC1 in two states (N=100). (F) Crosslinked lysine pairs in KIF5C-KLC1 were mapped onto the domain diagram.
Integrative modeling reveals molecular architecture of autoinhibited KIF5C-KLC1.
(A) The proposed model of autoinhibited KIF5C-KLC1 in cartoon diagram. (B) A model of KIF5C-KLC1(CC) via integrative modeling. The gray density is the low resolution map from negative staining EM. (C) The crosslinked pairs between KLC1-CC and KIF5C stalk, shown in the structural model. (D) The locations for the TPR domains in KIF5C-KLC1. (E) Crosslinked lysine pairs between TPR domains and KIF5C.
Disruption of the hierarchical folding activates KIF5B motility in vitro.
(A) The designed KIF5B mutations and truncations shown on the domain diagram. (B) Example kymographs of KIF5B with different truncations and the corresponding landing rate. Landing rate (Events/pm/s/pM): Lines show mean ± SEM: 1.07 ± 0.16 (full length), 14.92 ± 1.30 (1-909), 15.75 + 0.70(1-890), 26.87 ± 1.65 (1-565), 53.42 ± 3.47 (1-420). N=2; n=25, 20, 23, 15 and 14 MTs. One-way ANOVA followed by Dunnett’s test. ****P<0.0001. (C) Example kymographs of KIF5B with single mutation and the corresponding landing rate. Landing rate (Events/pm/s/pM): Lines show mean + SEM: 1.07 ± 0.16 (full ength), 3.93 ± 0.32 (CC2), 4.93 ± 0.42 (Hinge), 6.90 ± 0.78 (IAK), 0.67 + 0.32 (Linker). N=2; n=25, 25, 25, 20, and 9 MTs. One-way ANOVA followed by Dunnett’s test. *P<0.05, ***P<0.001, n.s not significant. (D) Example kymographs of KIF5B with combined mutations and the corresponding landing rate. Landing rate (Events/pm/s/pM): Lines show mean ± SEM: 1.07 ± 0.16 (full length), 6.44 + 0.40 (CC2+hinge), 7.66 ± 0.86 (CC2+IAK), 9.65 + 0.85 (Hinge+IAK), 10.49 ± 0.57 (CC2+hinge+IAK). N=2; n=25, 15, 12, 20, and 32 MTs. One-way ANOVA followed by Dunnett’s test. **P<0.01, ***P<0.001, ****P<0.0001.
TRAK1 N-terminus associates with KIF5B with the same location as KLC1 and reduces the crosslinks within KIF5B.
(A) Proposed model of TRAK1 activating KIF5B. (B) The size exclusion chromatography profile of TRAK1(1-395)-KIF5B complex and the corresponding SDS-PAGE analysis. (C) The mass photometry result for the TRAK1(1-395)-KIF5B complex. (D) The in vitro pull down assay between different KIF5B fragments and TRAK1(1-395). (E) The XL-MS results of TRAK1(1-395)-KIF5B.
