Figure 2. Simulations reveal opening of blebbistatin’s cryptic pocket.

(A) The distribution of pocket volumes from experimental crystal structures queried from the Protein Data Bank shows that the blebbistatin pocket is cryptic. The inset is a random selection of 15 structures from the accompanying distribution with an overlaid blebbistatin molecule in orange. All experimentally determined myosin structures display steric clash with a blebbistatin molecule aligned based on its contact residues in a blebbistatin-bound, or holo, structure (PDB: 1YV3). (B) Blebbistatin pocket volumes in simulations of fast skeletal myosin IIA reveal substantial pocket opening. The blebbistatin pocket volume from a ligand-bound crystal structure (PDB: 1YV3) is delineated by an orange vertical line in both panels. Simulated P(v) corresponds to the probability of adopting a given volume for each bin in the histogram. (C) MD simulations explore open holo-like states. Structure of an open conformation of the blebbistatin binding pocket from MD simulations reveals good structural alignment with the holo crystal structure (0.55 Å root mean square deviation of contact residue backbone heavy atom and Cβ positions). Blebbistatin is shown in orange with the pocket from the MD structure shown as a cyan contour. Selected residues in the blebbistatin pocket (Y269, L270, and F657) have the same backbone and sidechain positions as in the holo crystal structure. Note that reported pocket volumes are smaller than the space available to ligands because of an algorithm choice made to avoid erroneous detection of small pockets (see Materials and methods for details).

Figure 2—figure supplement 1. Trajectory traces for long simulations of skeletal muscle myosin reveal opening in all long MD trajectories.