Interaction between stretch of residues 633-642 (actin binding site) and nucleotide binding site on skeletal myosin subfragment 1 heavy chain

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Abstract
Using a complementary sequence or antipeptide to selectively neutralize the stretch of residues 633-642 of skeletal myosin heavy chain, we recently demonstrated that this segment is an actin binding site operating in the absence as in the presence of nucleotide and that this stretch 633-642 is not part of the nucleotide binding site [Chaussepied and Morales (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7471-7475]. In the present study, we determined that the covalent cross-linking of the antipeptide to the stretch 633-642 [induced by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide] does not alter the overall polypeptide conformation since no changes were observed on the far-ultraviolet CD spectra and thiol reactivity measurements. The presence of the antipeptide did not influence significantly the enhancement of tryptophan fluorescence induced by ATP.cntdot.Mg2+ or ADP.cntdot.Mg2+ binding to the myosin head (S1) nor did on the ATP.cntdot.Mg2+-induced tryptic proteolysis of S1 heavy chain. Moreover, fluorescence quenching studies, using acrylamide and the analogue, 1,N6-ethenoadenosine 5''-triphosphate, indicated that the nucleotide bound to antipeptide-S1 complex has an accessibility to the solute quencher close to that observed when it is bound to native S1. Additionally, neutralization of the stretch 633-642 of the S1 heavy chain by the antipeptide did not influence the stabilization of the Mg2+.cntdot.ADP.cntdot.sodium vanadate-S1 complex. On the other hand, experiments using antipeptide-induced protection against the cleavage of the S1 heavy chain by Arg-C protease demonstrated that the presence of Mg2+.cntdot.ADP.cntdot.sodium vanadate in the S1 nucleotide site did not affect the interaction of the antipeptide with the stretch of residues 633-642. Together, these results show that the occupancy of the stretch of residues 633-642 by the antipeptide does not affect the overall structure or the ATP binding and hydrolysis properties of skeletal myosin head and that the reactivity of the stretch 633-642 is not significantly dependent on the presence of nucleotide in the active site. Because the stretch 633-642 seems available to actin interaction in the presence of nucleotide and because blocking it strongly decreases actin interaction with S1-nucleotide complex, it is proposed that the stretch of residues 633-642 represents an essential constituent of the so-called "weak" actin-S1 interface.

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