Electron cryo-microscopy shows how strong binding of myosin to actin releases nucleotide

Abstract

Muscle contraction involves the cyclic interaction of the myosin cross-bridges with the actin filament, which is coupled to steps in the hydrolysis of ATP. While bound to actin each cross-bridge undergoes a conformational change, often referred to as the "power stroke", which moves the actin filament past the myosin filaments; this is associated with the release of the products of ATP hydrolysis and a stronger binding of myosin to actin. The association of a new ATP molecule weakens the binding again, and the attached cross-bridge rapidly dissociates from actin. The nucleotide is then hydrolysed, the conformational change reverses, and the myosin cross-bridge reattaches to actin. X-ray crystallography has determined the structural basis of the power stroke, but it is still not clear why the binding of actin weakens that of the nucleotide and vice versa. Here we describe, by fitting atomic models of actin and the myosin cross-bridge into high-resolution electron cryo-microscopy three-dimensional reconstructions, the molecular basis of this linkage. The closing of the actin-binding cleft when actin binds is structurally coupled to the opening of the nucleotide-binding pocket.

Myosin cleft movement and its coupling to actomyosin dissociation

“…1c,d). Excimers arise favours dimers with antiparallel rings, while the modelled rigor state, with a closed cleft 7 , favours parallel rings (Fig. 1c,d).…”

“…The rigor structure, as modelled by Holmes et al 7 , indicates that the α-carbon distance between residues 416 and 537 (Dd numbering) is 11 Å, compared to 17 to 19 Å in the apo and nucleotide-bound states of myosin in the absence of actin. Modelling shows that pyrene residues attached via acetamido-cysteine linkers can span these distances to form dimers without steric clashes with protein residues (Fig.…”

“…Initial docking attempts suggested that the cleft between the upper and lower 50K myosin domains might close on forming the rigor state 12 . This approach has been refined to the point of identifying a potential hinge point around switch 1 at the myosin nucleotide site that allows the upper 50K domain to rotate towards the lower 50K domain 7 . Recent crystallographic studies support this concept 6,8,9 .…”

Invited review: Probing the structures of muscle regulatory proteins using small‐angle solution scattering

“…The inherent flexibility within key muscle protein complexes has often frustrated X-ray crystallographic analyses (e.g., in obtaining crystals for the apo and Ca 21 -bound states of the Tn and Tn/Tm complexes), while NMR investigations are limited to relatively small, isolated components. Electron microscopy images have been exceptionally informative for probing the structures of huge macromolecular assemblies, [14][15][16] but they generally have insufficient resolution to resolve subunit boundaries and intersubunit distances. Small-angle solution scattering, when combined with the results of these more traditional structural biology tools, has provided important complementary information that has aided in advancing our understanding of muscle regulatory proteins.…”

Fluorescence labeling and computational analysis of the strut of myosin’s 50 kDa cleft

“…These are the P-loop that is a common feature of a large number of enzymes that bind nucleotide, the switch-1 loop and the switch -2 loop [96,97] be triggered by actin-binding cleft closure [35]. Further, pivoting the upper-50-kDa subdomain around a hinge located near the active site allows for greatly improved fitting of the skeletal myosin crystal structure to cryo-EM reconstructions of the acto-myosin complex [98]. Such a pivot would close the actin-binding cleft while opening the active site, as predicted by Rayment et al [35].…”