Myosin research

We are interested in the investigation of the mechanism of the communication between the functional regions of the myosin  motor domain. Here we present our most current results from this field.

I) The mechanism of the reverse recovery-step, phosphate release, and actin activation of Dictyostelium myosin II.

The rate-limiting step of the myosin basal ATPase (i.e., in absence of actin) is assumed to be a post-hydrolysis swinging of the lever-arm (reverse recovery-step), that limits the subsequent rapid product release steps. However, direct experimental evidence for this assignment is lacking. To investigate the binding and the release of ADP and phosphate independently from the lever-arm motion, two single tryptophan containing motor domains of Dictyostelium myosin II were used. The single tryptophans of the W129+ and W501+ constructs are located at the entrance of the nucleotide binding pocket and near the lever-arm, respectively. Kinetic experiments show that the rate-limiting step in the basal ATPase cycle is indeed the reverse recovery-step, which is a slow equilibrium step (k(forward)=0.05 s(-1), k(reverse)=0.15 s(-1)) that precedes the phosphate release step. Actin directly activates the reverse recovery-step which becomes practically irreversible in actin bound form, triggering the power-stroke. Even at low actin concentrations the power-stroke occurs in the actin-attached states despite the low actin affinity of myosin in the pre-power-stroke conformation.

II) Kinetic characterization of the function of myosin loop 4 in the actin-myosin interaction.

Myosin interacts with actin during its enzymatic cycle, and actin stimulates myosin’s ATPase activity. There are extensive interaction surfaces on both actin and myosin. Several surface loops of myosin play different roles in actomyosin interaction. However, the functional role of loop 4 in actin binding is still ambiguous. We explored the role of loop 4 by either mutating its conserved acidic group, Glu-365, to Gln (E365Q), or by replacing the entire loop with three glycines (DeltaAL) in a Dictyostelium discoideum myosin II motor domain (MD) containing a single tryptophan residue. This native tryptophan (Trp-501) is located in the relay loop and is sensitive to nucleotide binding and lever-arm movement. Fluorescence and fast kinetic measurements showed that the mutations in loop 4 do not alter the enzymatic steps of the ATPase cycle in the absence of actin. By contrast, actin binding was significantly weakened in the absence and presence of ADP and ATP in both mutants. Because the strength of actin-myosin interaction increases in the order of rigor, ADP, and ATP complex, we conclude that loop 4 is a functional actin-binding region that stabilizes actomyosin complex, particularly in weak actin-binding states.

III) Reversible movement of switch 1 loop of myosin determines actin interaction

The conserved switch 1 loop of P-loop NTPases is implicated as a central element that transmits information between the nucleotide-binding pocket and the binding site of the partner proteins. Recent structural studies have identified two states of switch 1 in G-proteins and myosin, but their role in the transduction mechanism has yet to be clarified. Single tryptophan residues were introduced into the switch 1 region of myosin II motor domain and studied by rapid reaction methods. We found that in the presence of MgADP, two states of switch 1 exist in dynamic equilibrium. Actin binding shifts the equilibrium towards one of the MgADP states, whereas ATP strongly favors the other. In the light of electron cryo-microscopic and X-ray crystallographic results, these findings lead to a specific structural model in which the equilibrium constant between the two states of switch 1 is coupled to the strength of the actin-myosin interaction. This has implications for the enzymatic mechanism of G-proteins and possibly P-loop NTPases in general.