The myosin (SH2-50-kilodalton fragment) cross-link: location and consequences

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Abstract
Some of us recently described a new interthiol cross-link which occurs in the skeletal myosin subfragment 1-MgADP complex between the reactive sulfhydryl group "SH2" (Cys-697) and a thiol (named SHx) of the 50-kilodalton (kDa) central domain of the heavy chain; this link leads to the entrapment of the nucleotide at the active site [Chaussepied, P., Mornet, D., and Kassab, R. (1986) Proc. Natl. Acad. Sci. U.S.A. 89, 2037-2041]. In the present study, we identify SHx as Cys-540 of the 50-kDa fragment. The portion of the heavy chain including this residue and also extending to Cys-522 that is cross-linkable to the "SH1" thiol [Ue, K. (1987) Biochemistry 26, 1889-1894] is near the SH2-SH1 region. Furthermore, various spectral and enzymatic properties of the (Cys697-Cys540)-N,N''-p-phenylenedimaleimide (pPDM)-cross-linked myosin chymotryptic subfragment 1 (S-1) were established and compared to those for the well-known (SH1.sbd.SH2).sbd.pPDM-cross-linked S-1. The circular dichroism spectra of the new derivative were similar to those of native S-1 complexed to MgADP. At 15 mM ionic strength, (Cys697.sbd.Cys540).sbd.S-1 binds very strongly to unregulated actin (Ka = 7 .times. 106 M-1), and the actin binding is very weakly affected by ionic strength. Joining actin with the (Cys697.sbd.Cys540).sbd.S-1 heavy chain, using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide, produces different species than does joining unmodified S-1 with actin. In contrast to the SH1.sbd.SH2-cross-linked S-1 which is thought to resemble the M.cntdot.ATP (M.cntdot.ADP.cntdot.Pi) state [Chalovich, J. M., Greene, L. E., and Eisenberg, E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4909.sbd.4913], the (Cys697.sbd.Cys540).sbd.S-1 conformation seems to simulate in M.cntdot.ADP state.