Probing the hydrophobic interactions in the skeletal actomyosin subfragment 1 and its nucleotide complexes by zero-length cross-linking with a nickel-peptide chelate

The complex of Ni(LT) and the tripeptide Gly-Gly-His catalyzes, in the presence of monoperoxyphthalic acid, a zero-length protein-protein cross-linking via an oxidative radical pathway involving mainly aromatic amino acids and not at all nucleophilic residues [Brown, K. C., Yang, S.-H., and Kodadek, T. (1995) Biochemistry 34, 4733-4739]. We have taken advantage of this unprecedented cross-linking system to directly and selectively probe the solution structure and functioning of the hydrophobic interface between F-actin and skeletal myosin subfragment 1 (S-l) at the level of its aromatic components, in the absence and in the presence of nuclotides (ATP and ADP) or nucleotide analogs (AMPPNP, PPi, and ADP . AlF4). Following verification of the structure of the Ni(II)-peptide chelate and of its oxidized active form by electrospray mass spectrometry, complexes of F-actin and S-l or proteolytic S-l derivatives and complexes of S-l and proteolytic F-actin derivatives were readily cross-linked under various controlled conditions without apparent alteration of the acto-S-l recognition. The covalent adducts were identified on electrophoretic gels using specific protein labeling with the oxidation-resistant fluorophor, monobromobimane, combined with immunochemical staining. Two types of actin-heavy chain conjugates were produced. One, with a mass of 180 kDa, was formed in the rigor state or with ADP bound; the other one, with a mass of 200 kDa, was generated from the ternary complexes comprising a gamma-P-containing ligand; They were accumulated with an efficiency of 8 and 6%, respectively. For each reversible complex, the 180 kDa:200 kDa band ratio was essentially as predicted from the nucleotide-dependent A to R equilibrium mechanism of the acto-S-l interaction in solution [Geeves, A. M., and Conibear, P. B. (1995) Biosphys. J. 68, 194s-201s]. Both covalent species resulted from the cross-linking of an actin monomer to the central 50 kDa segment, and their distinct mobilities reflect gamma-P-mediated structural changes at or near the actin-50 kDa fragment interface. Peptide mapping showed the cross-linking to take place between the 506-561 S-1 segment and the 48-113 actin stretch. The localization of these regions in the atomic F-actin-S-l model implies that nucleotide-modulated close contacts, involving aromatic residues, are operating between the C-terminal helix of the hydrophobic strong actin-binding motif of S-l bound to the primary actin monomer and the top portion of the adjacent lower actin subunit. The specificity of the nickel-peptide cross-linking, as assessed with the acto-S-l complex, makes it a candidate for potential general use in investigations of the hydrophobic interactions within other protein motor-based assemblies.