2.8 Å crystal structure of nucleotide-free G234V mutant kinesin. (A) Crystal packing of nucleotide-free G234V mutant. 2F_o−F_c map is shown in mesh (dark-blue), and the densities around the asymmetric unit are colored in pink. The proximal ends of the neck linkers of chains A and B face each other, with a cylindrical cavity between them. This cavity allows an antiparallel β-sheet formation between the two stretched neck linkers of chains A and B (shown in blue). While this demonstrates the neck linker peptides have an ability to form antiparallel β-sheets, such an arrangement would be geometrically impossible for the two neck linkers in a dimer, as their C termini are joined in parallel by the neck coiled-coil. In the G234A apo structure, we did not observe density corresponding to the antiparallel β-sheet in the cavity, likely due to its slightly smaller cavity size. (B) Comparison of the overall structures of nucleotide-free G234A (cyan) and G234V (yellow) crystal structures. G234V kinesin structure is nearly identical to that of G234A, except for the difference in the P-loop. (C) Close-up view of the nucleotide-binding pocket of G234A and G234V mutants (dotted rectangle in B). The side chains of residue 234 are shown as space-filling. The P-loop (pink) of G234V displayed a closed conformation, distinct from previously solved kinesin structures, indicating that the P-loop can adopt different configurations without bound phosphate. (D) Close-up view of the α4/α6 helices of nucleotide-free G234A (blue) and G234V (yellow) (the G234 structure is included from Fig. 1 C). The first two residues of the neck linker, K323 and T324, are highlighted as ball-and-stick models (G234A in green, G234V in red). In the G234V mutant, the T324 residue of the neck linker was visible and shifted toward the minus-end of the microtubule compared with that of G234A.