Phosphorylation-dependent structural changes in the regulatory light chaindomain of smooth muscle heavy meromyosin

Smooth muscle heavy meromyosin, a double-headed proteolytic fragment of myo sin lacking the COOH-terminal two-thirds of the tail, has been shown previo usly to be regulate by phosphorylation, To examine phosphorylation-dependen t structural changes near the head-tail junction, we prepared five well reg ulated heavy meromyosins containing single-cysteine mutants of the human sm ooth muscle regulatory light chain labeled with the photocross-linking reag ent, benzophenone-iodoacetamide, For those mutants that generated cross-lin ks, only one type of cross-linked species was observed, a regulatory light chain dimer, Irradiated mutants fell into two classes. First, for Q15C, A23 C, and wild type (Cys-108), a regulatory light chain dimer was formed for d ephosphorylated but not thiophosphorylated heavy meromyosin. These data pro vide direct chemical evidence that in the dephosphorylated state, Gln-15, A la-BS, and Cys-108 on one head are positioned near (within 8.9 Angstrom) th e regulatory light chain of the partner head and that thiophosphorylation a bolishes proximity, This behavior was also observed for the Q15C mutant on a truncated heavy meromyosin lacking both catalytic domains. For the actin- heavy meromyosin complex, cross-links were formed in both de- and thiophosp horylated states. S59C and T134C mutants were in a second mutant class, whe re regulatory light chain dimers were not detected in dephosphorylated or t hiophosphorylated heavy meromyosin, suggesting positions outside the region of interaction of the regulatory light chains.