Fig. 4: The MT-binding interface of Pbkinesin-8B-MD and contributions to motor function.

a Middle, ribbon depiction of the Pbkinesin-8B-MD and tubulin dimer NN state model, with Pbkinesin-8B-MD in green, α-tubulin in dark grey and β-tubulin in light grey; left, zoomed view of the Pbkinesin-8B-MD MT-binding surface coloured according to contacts with α-tubulin (dark grey) and β-tubulin (light grey); right, MT footprint of Pbkinesin-8B-MD on α- and β-tubulin indicated in dark green (tubulin residues <5 Å distance from the bound motor). Labels indicate the specific contacting secondary structure elements in tubulin dimer and Pbkinesin-8B-MD(in bracket). b Structural alignment of the Pbkinesin-8B-MD model (green) and KIF5B motor domain model (PDB 6OJQ, white), focusing on the loop 12, with the Pbkinesin-8B-MD-NN cryo-EM density shown in mesh representation. A sequence alignment of this region, and the swap mutant, is depicted below. PbKinesin-8B-MD loop 12 is coloured pink with positively charged residues coloured blue. Negatively charged residues in the adjacent H12 of β-tubulin are coloured red; c Structural alignment of the Pbkinesin-8B-MD model (green) and KIF5B motor domain model (PDB 6OJQ, white), focusing on loop 2, with the Pbkinesin-8B-MD-NN cryo-EM density shown in mesh. A sequence alignment of this region, and the sequence of the swap mutant, is depicted below. Pbkinesin-8B-MD loop 2 is coloured purple with positively charged residues coloured blue, negatively charged residues coloured red and hydrophobic residues coloured orange. Negatively charged residues in the adjacent H12 of α-tubulin are also coloured red, indicating the potential electrostatic interactions between loop 2 and the MT surface. d GMPCPP-MT-stimulated ATPase activity of Pbkinesin-8B-MD, Pbkinesin-8B-MD-L2^KIF5B and Pbkinesin-8B-MD-L12^KIF5B. Data (n = 3 for each point, mean ± SD) was fitted using Michaelis-Menten equation, from which the Kcat and K_M were calculated in Prism9; e Paclitaxel-stabilised-MT depolymerisation rate (nm/s) for Pbkinesin-8B-MD, Pbkinesin-8B-MD-L2^KIF5B and Pbkinesin-8B-MD-L12^KIF5B in the presence of ATP. Error bars represent the mean ± SD and individual measurements are also plotted. Ordinary one-way ANOVA was performed in Prism. Significance values are displayed as asterisks, ****p-values < 0.0001; ns not significant, p = 0.5029. N_{Pbkinesin-8B-MD} = 97 ends, N_{Pbkinesin-8B-MD-L2}^KIF5B = 86 ends, N_{Pbkinesin-8B-MD-L12}^KIF5B = 100 ends, N_{no-kinesin-8B} = 85 ends.