Structural changes induced in Ca2+-regulated myosin filaments Structural changes induced in Ca2+-regulated myosin filaments by Ca2+ and ATP by Ca2+ and ATP

We have used electron microscopy and proteolytic susceptibility to study the structural basis of myosin-linked regulation in synthetic filaments of scallop striated muscle myosin. Using papain as a probe of the structure of the head-rod junction, we find that this region of myosin is approximately five times more susceptible to proteolytic attack under activating (ATP/high Ca 2÷) or rigor (no ATP) conditions than under relaxing conditions (ATP/Iow Ca2+). A similar result was obtained with native myosin filaments in a crude homogenate of scallop muscle. Proteolytic susceptibility under conditions in which ADP or adenosine 5'-(~,3~-imidotriphosphate) (AMPPNP) replaced ATP was similar to that in the absence of nucleotide. Synthetic myosin filaments negatively stained under relaxing conditions showed a compact structure, in which the myosin cross-bridges were close to the filament backbone and well ordered, with a clear 14.5nm axial repeat. Under activating or rigor conditions, the cross-bridges became clumped and disordered and frequently projected further from the filament backbone, as has been found with native filaments; when ADP or AMPPNP replaced ATE the cross-bridges were also disordered. We conclude (a) that Ca 2÷ and ATP affect the affinity of the myosin cross-bridges for the filament backbone or for each other; (b) that the changes observed in the myosin filaments reflect a property of the myosin molecules alone, and are unlikely to be an artifact of negative staining; and (c) that the ordered structure occurs only in the relaxed state, requiring both the presence of hydrolyzed ATP on the myosin heads and the absence of Ca 2÷. C ONTRACTION of muscle is regulated via Ca2+-depen dent protein switches on the actin or myosin filaments or both, depending on the muscle and species. There are two basic forms of myosin-linked regulation. In one, found in vertebrate smooth and nonmuscle cells and in some invertebrate striated muscles, contraction is switched on by the Ca2+-dependent phosphorylation of the myosin regulatory light chains (Sobieszek, 1977; Sherry et al., 1978; Adelstein and Eisenberg, 1980; Kendrick-Jones and Scholey, 1981; Sellers, 1981). In the other, exemplified by the scallop and occurring in many invertebrate striated muscles, contraction is initiated when Ca 2÷ binds directly to the myosin heads (Kendrick-Jones et al., 1970; Szent-Gy6rgyi et al., 1973; Lehman and Szent-Gyfrgyi, 1975). The structural basis of myosin-linked regulation is not yet understood. In the case of the scallop, it has been established that the regulatory and essential light chains and the heavy chain are all involved in regulation (Szent-Gy6rgyi et al., 1973; Collins et al., 1986; Vibert and Cohen, 1988), forming a regulatory domain in the neck region of the myosin head (Szentkiralyi, 1984; Winkelmann et al., 1984; Bennett et al., 1984). Fluorescence measurements give evidence for local structural changes in the myosin heads when they bind Ca 2÷ (Wells et al., 1985) or ATP, and there is evidence that these changes involve movement of the light chains (Hardwicke et al., 1983; Hardwicke and Szent-Gyfrgyi, 1985; Wells et al., 1985). Large changes in the morphology of myosin molecules from vertebrate smooth muscle have been associated with the state of phosphorylation of the regulatory light chains (Craig et al., 1983; Trybus and Lowey, 1984; Ikebe and Hartshorne, 1984; Suzuki et al., 1985, 1988), and there is evidence that related changes may also occur in scallop myosin (Frado, L.-L. Y,, and R. Craig, manuscript in preparation). Evidence for regulatory changes in filament structure in both phosphorylationand direct Ca2+-dependent, myosinregulatory systems has come from ultrastructural studies. In the switched off (relaxed) state, myosin filaments of both types have a regular, ordered structure that becomes disordered when the filaments are activated (Ikebe and Ogihara, 1982; Vibert and Craig, 1985; Craig et al., 1987). However, the basis of this order-disorder transition is not understood. It remains possible that the observed changes could be artifacts of the negative-staining procedure or may have been caused by interaction of myosin cross-bridges with actin or other proteins in the crude filament homogenates used in some cases. In this paper we show that neither of these possibilities is likely: first by comparing the results of native filaments with those of synthetic filaments made from purified myosin, where the possibility of interaction with actin is © The Rockefeller University Press, 0021-9525/89/08/529/10 $2.00 The Journal of Cell Biology, Volume 109, August 1989 529-538 529 on July 4, 2008 w w w . jc.org D ow nladed fom Published August 1, 1989