Actin-binding cleft closure in myosin II probed by site-directed spin labeling and pulsed EPR

Abstract

We present a structurally dynamic model for nucleotide-and actin-induced closure of the actin-binding cleft of myosin, based on site-directed spin labeling and electron paramagnetic resonance (EPR) in Dictyostelium myosin II. The actin-binding cleft is a solventfilled cavity that extends to the nucleotide-binding pocket and has been predicted to close upon strong actin binding. Single-cysteine labeling sites were engineered to probe mobility and accessibility within the cleft. Addition of ADP and vanadate, which traps the posthydrolysis biochemical state, influenced probe mobility and accessibility slightly, whereas actin binding caused more dramatic changes in accessibility, consistent with cleft closure. We engineered five pairs of cysteine labeling sites to straddle the cleft, each pair having one label on the upper 50-kDa domain and one on the lower 50-kDa domain. Distances between spin-labeled sites were determined from the resulting spin-spin interactions, as measured by continuous wave EPR for distances of 0.7-2 nm or pulsed EPR (double electron-electron resonance) for distances of 1.7-6 nm. Because of the high distance resolution of EPR, at least two distinct structural states of the cleft were resolved. Each of the biochemical states tested (prehydrolysis, posthydrolysis, and rigor), reflects a mixture of these structural states, indicating that the coupling between biochemical and structural states is not rigid. The resulting model is much more dynamic than previously envisioned, with both open and closed conformations of the cleft interconverting, even in the rigor actomyosin complex.double electron-electron resonance ͉ dipolar interaction ͉ actomyosin M yosin motors use the energy derived from ATP hydrolysis to generate force for a diverse repertoire of biological tasks (1). Class II myosins produce muscle contraction and are involved in cytokinesis and cell motility. The present study focuses on Dictyostelium discoideum (Dicty) myosin II, which offers optimal facility for expression and purification of mutants, and is functionally similar to muscle myosin II (2, 3).

Structural kinetics of myosin by transient time-resolved FRET

“…For example, in the M Ã crystal structure, switch loop I and switch loop II of the active site do not appear to be in the proper position for catalysis of ATP hydrolysis (14,16), suggesting that step 3′ in Scheme 3 is not likely to occur. Indeed the present study, coupled with previous studies, indicates precisely that structural changes in the force-generating region (11,21) and the actin-binding cleft (22) are not tightly coupled to ligand binding at the active site. Rather, each crystal structure represents a trapped structural state among the much larger repertoire available to each biochemical state in solution (4).…”

“…The dependence of myosin ATPase activity on actin concentration was fitted to determine V max (activity at saturating actin) as reported in Table 2. As reported previously (11), both basal and actin-activated ATPase activities were comparable (within a factor of 2) between unlabeled and labeled proteins and were also comparable (within a factor of 2) to values reported for other Dictyostelium myosin constructs (20,22,33), indicating that neither mutations nor labeling caused significant effects on myosin catalytic activity (Table 2). ðTRÞ 2 FRET Experiments.…”

“…Change in the intrinsic fluorescence of a myosin-nucleotide analog complex upon pressure and temperature jumps suggests a transition between two structural states of myosin (17). Spectroscopic data on myosin with probes at the active site (18,19), in the forcegenerating region (11,20,21), or in the actin-binding cleft (22) show evidence for two resolved structural states (conformations) in the presence of a single biochemical state (bound ligand). TR-FRET has provided a clear structural picture of this phenomenon: With a single nucleotide analog bound to myosin, two distinct structural states (M Ã straight and M ÃÃ bent) were resolved for the entire head (23) (Fig.…”

Phosphate enhances myosin-powered actin filament velocity under acidic conditions in a motility assay

“…Thus the internal atomic motions within the active site that govern P i rebinding may be much more dynamic than the static high-resolution structures might imply. This notion is supported by recent observations that opening and closing of myosin's actin-binding cleft is highly dynamic in solution (17). Future work should attempt to capture the dynamic nature of P i release and rebinding within myosin's active site to provide a more detailed picture of how P i is coupled to the generation of force and motion during contraction.…”

Refolding of the integral membrane protein light-harvesting complex II monitored by pulse EPR

“…Doubleelectron-electron resonance (DEER) spectroscopy allows the measurement of distances between two spin-labeled residues in the range of 2-6 nm, in favorable cases even 8 nm (9, 10). This technique is especially useful for the prediction of protein structure (11, 12) as well as for measuring structure dynamics (13,14). Electron spin echo envelope modulation (ESEEM) spectroscopy, also a pulse EPR technique, yields additional structural information by determining the water accessibility of singly spin-labeled protein domains (15).…”