The effects of mutations in an actin-binding surface loop of myosin (loop 2) are described. Part of loop 2, the segment between myosin residues 618 and 622, was replaced with sequences enlarged by the introduction of positively charged GKK or neutral GNN motifs. Constructs with loops carrying up to 20 additional amino acids and charge variations from -1 to +12 were produced. Steady-state and transient kinetics were used to characterize the enzymatic behavior of the mutant motor domains. Binding of nucleotide was not affected by any of the alterations in loop 2. In regard to their interaction with actin, constructs with moderate charge changes (-1 to +2) displayed wild-type-like behavior. Introduction of more than one GKK motif led to stronger coupling between the actin- and nucleotide-binding sites of myosin and an up to 1000-fold increased affinity for actin in the absence of ATP and at zero ionic strength. In comparison to the wild-type construct M765, constructs with 4-12 extra charges displayed an increased dependence on ionic strength in their interaction with actin, a 2-3-fold increase in kcat, a more than 10-fold reduction in Kapp for actin, and a 34-70-fold increase in catalytic efficiency.

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We combined protein engineering and single molecule measurements to directly record the step size of a series of myosin constructs with shortened and elongated artificial neck domains. Our results show that the step size has a clear linear dependence on the length of the neck domain and we also established that mechanical amplification in the myosin motor is based on a rotation of the neck domain relative to the actin-bound head. For all our constructs, including those with artificial necks, the magnitude of the neck rotation concurrent with the displacement step was approximately 30 degrees. The engineered change in the step size of myosin marks a significant advance in our ability to selectively modify the functional properties of molecular motors.

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...