Stabilization of the actomyosin complex by negative charges on myosin

Sequence comparisons of members of the myosin superfamily show a high degre e of charge conservation in a surface exposed helix (Dictyostelium discoide um myosin II heavy chain residues S510 to K546). Most myosins display a tri plet of acidic residues at the equivalent positions to D. discoideum myosin II residues D530, E531, and Q532. The high degree of charge conservation s uggests 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) Biochemisty 36, 8465 -8473]. Here, we used steady-state and transient kinetics to characterize t he enzymatic competence of mutant constructs E531Q and Q532E, and their pro perties were compared with those of a loop 2 mutant with a 20 amino acid in sertion containing 12 positive charges (20/+12) [Furch et al. (1998) Bioche mistry 37, 6317-6326], double mutant Q532E(20/+12), and the native motor do main constructs. Our results confirm that charge changes at residues 531 an d 532 primarily affect actin binding with little change being communicated to the nucleotide pocket. Mutation D531Q reduces actin affinity (K-A) 10-fo ld, while Q532E leads to a 5-fold increase. The observed changes in K-A Ste m almost exclusively from variations in: the dissociation rate constant (k( -A)), with the introduction of a single negative charge at position 532 hav ing the same effect on k(-A) as the introduction of 12 positive charges in the loop 2 region.