Peptide mimetics of an actin-binding site on myosin span two functional domains on actin

Abstract

THE sites on the myosin heavy chain that interact with actin and are responsible for force generation are ill-defined: crosslinking^1,2 and experiments with isolated domains of the myosin head^3,4 implicate regions in both the 50K and 20K (molecular weights in thousands) domains of the myosin head (subfragment 1, S1) in this process. We have synthesized peptides from the sequence around the fast-reacting SH₁ thiol residue in the 20K domain of SI in order to delineate precisely an actin-binding site. We used a combination of ¹H-NMR and enzyme inhibition assay and also assessed the effects of peptides on skinned rabbit psoas muscle fibres to show that the region of amino acids 690–725 contains an actin-binding site. Peptides from this region bind to actin, act as mixed inhibitors of the actin-stimulated S1 Mg²⁺-ATPase, and influence the contractile force developed in skinned fibres, whereas peptides flanking this sequence are without effect in our test systems. Remarkably, peptides from the N-terminal half of this segment 690–725 increase force development in skinned fibres at submaximal activating concentrations of Ca²⁺, that is, they behave as calcium-sensitizers; C-terminal peptides, however, inhibit force development without effecting sensitivity to calcium. These different responses indicate that this region is probably binding at two functionally distinct sites on actin.