Functional characterization of the secondary actin binding site of myosin II

The role of the interaction between actin and the secondary actin binding s ite of myosin (segment 565-579 of rabbit skeletal muscle myosin, referred t o as loop 3 in this work) has been studied with proteolytically generated s mooth and skeletal muscle myosin subfragment 1 and recombinant Dictyosteliu m discoideum myosin II motor domain constructs. Carbodiimide-induced cross- linking between filamentous actin and myosin loop 3 took place only with th e motor domain of skeletal muscle myosin and not with those of smooth muscl e or D. discoideum myosin II. Chimeric constructs of the D. discoideum myos in motor domain containing loop 3 of either human skeletal muscle or nonmus cle myosin were generated, Significant actin cross-linking to the loop 3 re gion was obtained only with the skeletal muscle chimera both in the rigor a nd 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 t o 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 in crease in their actin dissociation constant, due to a significant increase in the rate for actin dissociation (k(-A)) with no significant change in th e rate for actin binding (k(+A)) The actin-activated ATPase activity was no t affected by the substitutions in the chimeric constructs. These results s uggest that actin interaction with the secondary actin binding site of myos in is specific for the loop 3 sequence of striated muscle myosin isoforms b ut is apparently not essential either for the formation of a high affinity actin-myosin interface or for the modulation of actomyosin ATPase activity.