Stabilization of the actomyosin complex by negative charges on myosin.
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. discoideummyosin 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.
Furch
M
M
Max-Planck-Institute for Medical Research, Department of Biophysics, Jahnstrasse 29, D-69120 Heidelberg, Germany.
Remmel
B
B
Geeves
M A
MA
Manstein
D J
DJ
D000199:Actins / Q000378:metabolism; D000205:Actomyosin / Q000737:chemistry* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D004789:Enzyme Activation / Q000235:genetics; D007700:Kinetics; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015879:Myosin Subfragments / Q000235:genetics / Q000378:metabolism; D009218:Myosins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011817:Rabbits; D055672:Static Electricity
D000199:Actins; D020409:Molecular Motor Proteins; D015879:Myosin Subfragments; D000255:Adenosine Triphosphate; D000205:Actomyosin; D009218:Myosins