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

Abstract

The complex of Ni(II) 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-1) at the level of its aromatic components, in the absence and in the presence of nucleotides (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-1 or proteolytic S-1 derivatives and complexes of S-1 and proteolytic F-actin derivatives were readily cross-linked under various controlled conditions without apparent alteration of the acto-S-1 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-1 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-1 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-1 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-1 complex, makes it a candidate for potential general use in investigations of the hydrophobic interactions within other protein motor-based assemblies.