title: 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 Cys(108) 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 Cys(108) from one regulatory light chain cross-linked to (71)GMMSEAPGPIN(81), a loop in the N-terminal half of the regulatory light chain, and to (4)RAKAKTTKKRPQR(16), 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.
authors.affiliation: Department of Biochemistry, University of Nevada, Reno, Nevada 89557, USA.
authors.forename: Jan L
authors.initials: JL
authors.lastname: Wahlstrom
authors.forename: M Allen
authors.initials: MA
authors.lastname: Randall
authors.forename: J David
authors.initials: JD
authors.lastname: Lawson
authors.forename: Derek E
authors.initials: DE
authors.lastname: Lyons
authors.forename: William F
authors.initials: WF
authors.lastname: Siems
authors.forename: Greg J
authors.initials: GJ
authors.lastname: Crouch
authors.forename: Regina
authors.initials: R
authors.lastname: Barr
authors.forename: Kevin C
authors.initials: KC
authors.lastname: Facemyer
authors.forename: Christine R
authors.initials: CR
authors.lastname: Cremo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D003432:Cross-Linking Reagents; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000378:metabolism; D015879:Myosin Subfragments / Q000737:chemistry* / Q000378:metabolism; D010446:Peptide Fragments; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
chemical_list: D003432:Cross-Linking Reagents; D015879:Myosin Subfragments; D010446:Peptide Fragments