Fig. 3: A two-step mechanism for MT-stimulated ADP release in Kif5b is supported by cryo-EM and MD simulations.

a, b Superimpositions of Kif5b-ADP without MTs (PDB code 1bg2, semi-transparent model) with our Kif5b^MoNeXXX ADP-state (opaque model) highlighting the proposed a Mg²⁺-release and b ADP destabilization mechanisms. Hydrogen bonds in the Kif5b-ADP state without and with MTs are shown as gray and black dashed lines, respectively. In (a), the movement of R190 and D231 α-carbon atoms is illustrated with black arrows. c Representative co-sedimentation assay of MT and Kif5b^MoNeXXX co-incubated with increasing amounts of MgCl₂ as indicated. Co-sedimentation was performed with taxol-stabilized MTs at 2 μM (tubulin dimer) and 0.5 μM Kif5b^MoNeXXX. P = Pellet, S = Supernatant. Molecular weights are indicated. d Densitometric quantification of Coomassie-stained bands in the SDS-PAGE analysis of co-sedimentation assays (as in c), showing the percentage of total Kif5b^MoNeXXX in pellet vs. supernatant fractions. n = 4 independent experiments for each condition. Data are presented as mean values ± SD. Source data are provided as a Source Data file. e Average standard deviation for the atomic distances used to monitor ADP stability within the binding pocket during three independent 1 μs MD replicas. Error bars are s.e.m. values. Source data are provided as a Source Data file. The MT-free/Mg²⁺-bound system is Kif5b-ADP without MTs (PDB code 1bg2). The MT-bound/Mg²⁺-free system is modeled based on our Kif5b^MoNeXXX ADP-state. f 2D distribution of two of the reaction coordinates: RMSD(ADP) and distance between its phosphorus Pβ and S98, monitored in the MD runs for the MT-free/Mg²⁺-bound system and g representative snapshot. Atom pair distances reported in (e) are shown as broken black lines. h, i like (f, g) for the MT-bound/Mg²⁺-free system. Compared to the MT-free/Mg²⁺-bound system, here loop 9 moves closer to the active site. j, k like (h, i) for Level 2 replicas. l, m like (h, i) for Level 3 replicas, leading to ADP dissociation. n Cartoon representation highlighting the loop 9 dynamics in the MT-free/Mg²⁺-bound system (dark yellow) and in the MT-bound/Mg²⁺-free system (yellow). In the latter system, there is a shift toward the active site that enables ADP displacement, as shown in (m). Source data for panels (f, h, j, l) are provided as a Source Data file.