SPECIFIC CROSS-LINKING OF THE SH1 THIOL OF SKELETAL MYOSIN SUBFRAGMENT-1 TO F-ACTIN AND G-ACTIN

Recently, we reported that (maleimidobenzoyl)-G-actin (MBS-G-actin), which was resistant to the salt and myosin subfragment 1 (S-1) induced polymerizations, reacts reversibly and covalently in solution with the S-1 heavy chain at or near the strong F-actin binding region [Bettache, N., Bertrand, R., & Kassab, R. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6028-6032]. Here, we have readily converted the MBS-G-actin into MBS-F-actin in the presence of phalloidin and salts. The binding of S-1 to the two actin derivatives carrying on their surface free reactive maleimidobenzoyl groups was investigated comparatively in cross-linking experiments performed under various conditions to probe further the molecular structure of the actin-heavy chain complex before and after the polymerization process. Like MBS-G-actin, the isolated MBS-F-actin, which did not undergo any intersubunit cross-linking, bound stoichiometrically to S-1, generating two kinds of actin-heavy chain covalent complexes migrating on electrophoretic gels at 180 and 140 kDa. The relative extent of their production was essentially dependent on pH for both G-and F-actins. At pH 8.0, the 180-kDa species was predominant, and at pH 7.0, the amount of the 140-kDa adduct increased at the expense of the 180-kDa entity. The cross-linking of MBS-F-actin to S-1 led to the superactivation of the MgATPase substantiating the ability of this derivative to stimulate the S-1 ATPase as the native protein. The 140-kDa complex was suppressed by blocking Cys-707 (SH1) in S-1 but not at all by the specific modification of Cys-697 (SH2). The addition of Mg (1 mM) increased selectively the yield of the 140-kDa product with both native S-1 and SH2-blocked S-1. The cross-linking between the MBS-actins and SHI-modified S-1 in the presence of MgADP did not yield the 140-kDa species. The cross-linking of the MBS-actins to fluorescently labeled split S-1 showed the conjugation of actin to the 50-kDa fragment in the 180-kDa species and to the 20-kDa fragment in the 140-kDa derivative. The data suggest that in the G- and F-MBS-actin-S-1 complexes the cross-linkable lysine side chain on actin to which the maleimidobenzoyl arm was attached is within 0.9-1.0 nm from two different S-1 heavy-chain segments, one of which includes the SHI thiol; these may be spatially related, forming together an unique actin recognition site in S-1.