FIGURE 2.

Nucleotide binding pocket in the nucleotide-free structures of wild type myosin VI (A), Δ-insert-1 mutant (B), and L310G mutant (C). Three nucleotide binding elements compose the active site of myosin: P-loop (green) from the N-terminal subdomain that binds the phosphate moiety of the nucleotide, Switch I (purple) from the U50 subdomain involved in the binding of Mg²⁺ and the γ-phosphate when nucleotide binds strongly, and Switch II (pink) that plays an important role for hydrolysis of the nucleotide. The ATP molecule (modeled in these structures) enters in the active site of these nucleotide-free molecules and binds via interactions with the P-loop (phosphate moiety) and the N-terminal subdomain (adenine ring). On the right in these panels, part of the U50 subdomain close to the active site is represented. Insert-1 (residues Cys-278–Ala-303) is represented in orange and is deleted in the Δ-insert-1 mutant. A, in wild type myosin VI, its presence repositions the loop (yellow) that precedes helix HK and notably positions Leu-310 close to the ATP molecule. B, in the Δ-insert-1 mutant, deletion of the 26-residue insert repositions Leu-310 (yellow) far from the nucleotide binding pocket, and the active site is much wider in this mutant. The length of the remaining loop that contains Leu-310 is identical to that found in other myosin such as myosin V. The main chain conformation of the loop differs, however, from that found in myosin V and six residues in the middle of the loop (Gly-277 linked to Gly-304–Ser-305–Leu-306–Lys-307) are too disordered in the structure to be modeled. C, in the L310G mutant, substitution of the leucine by glycine does not introduce any significant changes in the conformation of this loop or in insert-1. The only difference in this mutant is the absence of the side chain of Leu-310, which protrudes within the nucleotide binding pocket in the wild type structure, shown in white in this panel. To illustrate how the leucine side chain may interfere with different conformations explored by the nucleotide upon binding, an ATP molecule in the conformation found in a scallop structure (PDB 1S5C), in which nucleotide is partially bound via the adenine ring only, is represented in blue.