Stabilization of the Actomyosin Complex by Negative Charges on Myosin

Abstract

Sequence comparisons of members of the myosin superfamily show a high degree of charge conservation in a surface exposed helix (Dictyostelium discoideum myosin II heavy chain residues S510 to K546). Most myosins display a triplet of acidic residues at the equivalent positions to D. discoideum myosin II residues D530, E531, and Q532. The high degree of charge conservation suggests strong evolutionary constrain and that this region is important for myosin function. Mutations at position E531 were shown to strongly affect actin binding [Giese, K. C., and Spudich, J. A. (1997) Biochemistry 36, 8465-8473]. Here, we used steady-state and transient kinetics to characterize the enzymatic competence of mutant constructs E531Q and Q532E, and their properties were compared with those of a loop 2 mutant with a 20 amino acid insertion containing 12 positive charges (20/+12) [Furch et al. (1998) Biochemistry 37, 6317-6326], double mutant Q532E(20/+12), and the native motor domain constructs. Our results confirm that charge changes at residues 531 and 532 primarily affect actin binding with little change being communicated to the nucleotide pocket. Mutation D531Q reduces actin affinity (K A ) 10-fold, while Q532E leads to a 5-fold increase. The observed changes in K A stem almost exclusively from variations in the dissociation rate constant (k -A ), with the introduction of a single negative charge at position 532 having the same effect on k -A as the introduction of 12 positive charges in the loop 2 region.Myosin II drives muscle contraction and motile processes such as cytokinesis and cell motility. The ATP-dependent 1 interaction of the myosin head with actin is central to myosin driven motile processes. Atomic models of the actomyosin rigor complex show the interface between myosin and actin to consist of four major contact regions, suggesting a sequential mechanism of binding (1, 2). A first contact involves a highly charged, lysine-rich loop on myosin (loop 2) formed by residues S619 to V630 in the case of Dictyostelium discoideum myosin II and a cluster of acidic residues at the N-terminus of actin (3-6). A second contact region involves a helix-loop-helix structure formed by residues S510 to K546 (unless otherwise stated amino acid residues are numbered according to the D. discoideum myosin II sequence). A loop formed by residues L399 to V411 makes a third contact. These three contacts involve a single actin monomer and form the primary actin-binding site of myosin. In addition a loop protruding between residues L547 and H572 may reach the neighboring actin monomer one actin helix turn below (Figure 1).Previously, we have shown that the light-chain-binding domain (LCBD) plays no major role in the biochemical behavior of the myosin (7-9). Recombinant constructs without LCBD can be produced and purified in large amounts and are ideally suited for systematic studies of the structure, kinetics and function of the myosin motor. Therefore, we used construct M765, corresponding to the first 765 residues of D. disc...

Myosin 3A Kinase Activity Is Regulated by Phosphorylation of the Kinase Domain Activation Loop

“…Kinases are known to prefer phosphorylation of less well ordered and surface-exposed regions of proteins (17), which is consistent with studies that demonstrate loop 2 is highly disordered in myosins (46). Studies have demonstrated that the degree of positive charge in loop 2 can impact actin-activated ATPase activity and actin affinity of myosins (47)(48)(49). The additional negative charges associated with phosphorylation are well positioned to reduce actin affinity and ATPase activity.…”

Structural Model of Weak Binding Actomyosin in the Prepowerstroke State

“…We also confirm the interaction of loop 3 with two actin monomers (34,35), which are among the initial weak actin-binding interactions. Structural and mutational studies indicate that dynamic interactions of loop 2 with different regions of actin in the different states of actomyosin reflect its role in the weak to strong actin-binding transition of myosin (36,37). We found that loop 2 positioned differently in rigor compared with the weak actinbinding state.…”

Kinetic Characterization of Nonmuscle Myosin IIB at the Single Molecule Level

“…a high stiffness, no power stroke state). Such interactions were first detected in relaxed skinned muscle fibers which were oscillated at high frequency (53) and were later proposed to arise from nonspecific ionic interactions between loop II of myosin with actin (54). Such interactions were predicted to be the basis for the ability of unphosphorylated smooth muscle myosin to retard the movement of actin filaments by phosphorylated smooth muscle myosin in in vitro actin gliding motility assays (55).…”