Functional characterization of the secondary actin binding site of myosin II.
The role of the interaction between actin and the secondary actin binding site of myosin (segment 565-579 of rabbit skeletal muscle myosin, referred to as loop 3 in this work) has been studied with proteolytically generated smooth and skeletal muscle myosin subfragment 1 and recombinant Dictyostelium discoideum myosin II motor domain constructs. Carbodiimide-induced cross-linking between filamentous actin and myosin loop 3 took place only with the motor domain of skeletal muscle myosin and not with those of smooth muscle or D. discoideum myosin II. Chimeric constructs of the D. discoideum myosin motor domain containing loop 3 of either human skeletal muscle or nonmuscle myosin were generated. Significant actin cross-linking to the loop 3 region was obtained only with the skeletal muscle chimera both in the rigor and in the weak binding states, i.e., in the absence and in the presence of ATP analogues. Thrombin degradation of the cross-linked products was used to confirm the cross-linking site of myosin loop 3 within the actin segment 1-28. The skeletal muscle and nonmuscle myosin chimera showed a 4-6-fold increase in their actin dissociation constant, due to a significant increase in the rate for actin dissociation (k(-)(A)) with no significant change in the rate for actin binding (k(+A)). The actin-activated ATPase activity was not affected by the substitutions in the chimeric constructs. These results suggest that actin interaction with the secondary actin binding site of myosin is specific for the loop 3 sequence of striated muscle myosin isoforms but is apparently not essential either for the formation of a high affinity actin-myosin interface or for the modulation of actomyosin ATPase activity.
Van Dijk
J
J
Centre de Recherche de Biochimie Macromoléculaire du Centre National de la Recherche Scientifique, Institut Féderatif de Recherche 24, Montpellier, France.
Furch
M
M
Lafont
C
C
Manstein
D J
DJ
Chaussepied
P
P
D000199:Actins / Q000737:chemistry* / Q000378:metabolism*; D000205:Actomyosin / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002645:Chickens; D006801:Humans; D006868:Hydrolysis; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000378:metabolism; D018482:Muscle, Skeletal / Q000737:chemistry; D009130:Muscle, Smooth / Q000737:chemistry; D009218:Myosins / Q000737:chemistry* / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D020033:Protein Isoforms / Q000378:metabolism; D011817:Rabbits
D000199:Actins; D020409:Molecular Motor Proteins; D010446:Peptide Fragments; D020033:Protein Isoforms; D000255:Adenosine Triphosphate; D000205:Actomyosin; D009218:Myosins