Structural Model of the Regulatory Domain of Smooth Muscle Heavy Meromyosin

Abstract The goal of this study was to provide structural information about the regulatory domains of double-headed smooth muscle heavy meromyosin, including the N terminus of the regulatory light chain, in both the phosphorylated and unphosphorylated states. We extended our previous photo-cross-linking studies (Wu, X., Clack, B. A., Zhi, G., Stull, J. T., and Cremo, C. R. (1999)J. Biol. Chem. 274, 20328–20335) to determine regions of the regulatory light chain that are cross-linked by a cross-linker attached to Cys108 on the partner regulatory light chain. For this purpose, we have synthesized two new biotinylated sulfhydryl reactive photo-cross-linking reagents, benzophenone, 4-(N-iodoacetamido)-4′-(N-biotinylamido) and benzophenone, 4-(N-maleimido)-4′-(N-biotinylamido). Cross-linked peptides were purified by avidin affinity chromatography and characterized by Edman sequencing and mass spectrometry. Labeled Cys108 from one regulatory light chain cross-linked to71GMMSEAPGPIN81, a loop in the N-terminal half of the regulatory light chain, and to4RAKAKTTKKRPQR16, a region for which there is no atomic resolution data. Both cross-links were to the partner regulatory light chain and occurred in unphosphorylated but not phosphorylated heavy meromyosin. Using these data, data from our previous study, and atomic coordinates from various myosin isoforms, we have constructed a structural model of the regulatory domain in an unphosphorylated double-headed molecule that predicts the general location of the N terminus. The implications for the structural basis of the phosphorylation-mediated regulatory mechanism are discussed.