Photochemical cross‐linking of the skeletal myosin head heavy chain to actin subdomain‐1 at Arg95 and Arg28

Abstract

F-actin specifically substituted with the photocross-linker, p-azidophenylglyoxal, at Arg95 and Arg28 was isolated and characterized. Upon complexation with myosin subfragment-1 (Sl) and photolysis at 365 nm, it was readily cross-linked to the S1 heavy chain with a yield of about 13-25 %, generating four major actin-heavy-chain adducts with molecular masses in the range 165-240 kDa. The elevated Mg2'-ATPase of the covalent complexes displayed a turnover rate of 33 ? 8 s-I which is similar to the values reported earlier for other acto-S1 conjugates. The crosslinking between various proteolytic S1 and actin derivatives, combined with the fluorescent and immunochemical detection of the photocross-linked products, indicated that the arylnitrene group on Arg95 was inserted predominantly in the central 50-kDa region, whereas that attached to Arg28 mediated the selective cross-linking of the COOH-terminal 22-21-kDa fragments of the heavy chain, most probably by reacting at or near the connector segment between the 50-kDa and 20-kDa fragments. The rapid photoactivation and cross-linking to S1 of the substituted F-actin, which can be accomplished on a millisecond time scale, may serve to probe the structural dynamics of the interaction of the S1 heavy chain with subdomain-1 of actin during the ATPase cycle.During muscle contraction, the cyclic interactions between F-actin and the myosin heads or myosin subfragment-1 (Sl) are essential for the conversion of ATP hydrolysis into contractile force by the actomyosin-ATP complex. The nature of the nucleotide intermediates bound to the ATPase site is thought to determine the molecular movements and attitudes of the myosin heads over the actin filament (Botts et al., 1984;Huxley and Kress, 1985;Vibert and Cohen, 1988). The changes in the structure, localization and affinity of the binding sites at the actin-S1 interface would, therefore, play a major role in the transients of the displacement of S1 along F-actin.At present, it can be postulated that the generation of any acto-S 1 complex involves the specific association between the subdomain-1 of actin, the outermost domain of the actin protomere in the filament, containing all the known putative S1 interactive sites (Holmes et al., 1990;Milligan et al., 1990), and polypeptide stretches of the 20-kDa and 50-kDa fragments of the S1 heavy chain, the two potent actin-binding regions so far identified in the native protein (Mornet et Correspondence to P. Chaussepied or R.

Isolation of 88F Actin Mutants of and Possible Alterations in the Mutant Actin Structures

“…In Ifm(3)1 and Ifm(3)2 (both were named as act88F 1 by Lindsley & Zimm, 1992; but our record showed that they were independently isolated), Arg28 is changed to cysteine . Arg28 seems to be involved in the binding of actin to S1 according to the results of chemical cross-linking and proton NMR studies, and from the actin crystal structure (Bertrand et al, 1988;Moir et al, 1987;Kabsch et al, 1990;Van Eyk & Hodges, 1991;Bonafé et al, 1993). Abnormalities characteristic of R95C, such as myofibril degeneration and IFM degradation are also observed in these mutants (Sakai et al, 1991).…”

“…If this is the case, the lesion arising from an R95C substitution should result from improper interaction with other molecules. According to NMR and chemical cross-linking studies, Arg95 is considered to belong to the central contact region at which actin interacts with myosin S1 (Moir & Levine, 1986;Bertrand et al, 1988;Bonafé et al, 1993), although elucidation of the structure of myosin S1 and the actin-myosin complex failed to reveal the direct site of contact of Arg95 with myosin S1 (Rayment et al, 1993a,b;Schrö eder et al, 1993) . The possibility also remains that this site is altered actin interact with the heat shock response machinery.…”

Differences in the ionic interaction of actin with the motor domains of nonmuscle and muscle myosin II

“…Densitometric analysis of the Coomassie blue‐stained gels was carried out with a Shimadzu CS 930 high‐resolution gel scanner equipped with a computerized integrator. Western blot analysis using rabbit polyclonal antibodies directed against M765 or mouse monoclonal antipolyhistidine tag was performed as described [32].…”

Two identical hydrophobic clusters are present on the same actin monomer: interaction between one myosin subfragment‐1 and two actin monomers

“…~11 of these contacts involve a single monomer. A second mon-~,mer is close to the myosin heavy chain and the interaction i ~cludes the cz-helix formed by actin residues Trp79 to Asn92 flat formed a weak binding contact and was cross-linked [5,6].…”

“…~11 of these contacts involve a single monomer. A second mon-~,mer is close to the myosin heavy chain and the interaction i ~cludes the cz-helix formed by actin residues Trp79 to Asn92 flat formed a weak binding contact and was cross-linked [5,6].The Taylor-Amos model [7] of acto-S1 suggests that S1 has n extensive contact with an actin monomer (acl) and a weaker ,. ontact with an adjacent actin monomer (ac2); and two differ-,, nt rigor complexes were suggested [8,9], with SI binding to :-actin occurring in two steps with actin monomers.…”