Fig. 4: Kap3 makes a multipartite interaction with Kif3AB that permits autoinhibition.
From: Regulation of kinesin-2 motility by its β-hairpin motif
a, Size-exclusion chromatogram of reconstituted Kif3AB–Kap3 complex (red); schematics are also shown. Normalized Kif3AB and Kap3 traces are shown for comparison (dashed orange and yellow lines respectively). b, SDS–PAGE of the peak size-exclusion chromatography fraction. c, Top, composite TIRF image of the 488-nm channel (MT) and 640-nm channel (Alexa-Fluor-647-labeled Kif3A), offset in the y axis by 8 pixels, and the 561-nm channel (TMR-labeled-Kap3), offset in the y axis by 16 pixels. The assay was conducted with 1.5 nM Kif3AB, 7.5 nM Kap3 and 1 mM ATP. The presence of Kap3 does not activate Kif3AB microtubule binding or motility. Bottom, the same experiment as above, but with 1 mM AMPPNP. The assay was performed with 1 nM Kif3AB and 1 nM Kap3. Kif3AB and Kap3 colocalize in a complex. d, Top, example class averages of the Kif3AB–Kap3 complex, with features labeled. Bottom, corresponding AlphaFold3 (AF) model projections. e, Ribbon representation of the Kif3AB–Kap3 AlphaFold3 model. Protruding coiled-coil regions of Kif3AB shown in white. Further analysis of coiled-coil regions in this model shown in Extended Data Fig. 5. f, Close-up of the composite binding interface between Kap3 and Kif3AB C-terminal region in the AlphaFold3 model. The three main interfaces are labeled. Kif3AB β-hairpins are not occluded by Kap3 binding. g, Plot of the fraction of TMR-labeled Kap3 colocalizing with Alexa-Fluor-647-labeled Kif3AB for each indicated construct: ΔInt3 (Kif3A–Kif3BΔC90), ΔInt2+3 (Kif3A–Kif3BΔC114), ΔInt1 (Kif3AΔC89–Kif3B) and ΔInt1–3 (Kif3AΔC103–Kif3BΔC156). Measurements were taken from three technical replicates. Gray circles, average from each replicate. Lines, mean ± s.e.m. WT, n = 49, 68 and 81; ΔInt3, n = 54, 75 and 85; ΔInt2+3, n = 46, 70 and 82; ΔInt1, n = 68, 44 and 56; ΔInt1–3, n = 59, 58 and 28 molecules analyzed per replicate. Mean values for WT, ΔInt2+3, ΔInt1 and ΔInt1–3 are significantly different from each other (P < 0.01, one-way ANOVA followed by Tukey’s multiple comparison test). Exact P values are given in the Methods.
