title: The N-terminal lobes of both regulatory light chains interact with the tail domain in the 10 S-inhibited conformation of smooth muscle myosin.
abstract: In the presence of ATP, unphosphorylated smooth muscle myosin can form a catalytically inactive monomer that sediments at 10 Svedbergs (10 S). The tail of 10 S bends into thirds and interacts with the regulatory domain. ADP-P(i) is "trapped" at the active site, and consequently the ATPase activity is extremely low. We are interested in the structural basis for maintenance of this off state. Our prior photocross-linking work with 10 S showed that tail residues 1554-1583 are proximal to position 108 in the C-terminal lobe of one of the two regulatory light chains ( Olney, J. J., Sellers, J. R., and Cremo, C. R. (1996) J. Biol. Chem. 271, 20375-20384 ). These data suggested that the tail interacts with only one of the two regulatory light chains. Here we present data, using a photocross-linker on position 59 on the N-terminal lobe of the regulatory light chain (RLC), demonstrating that both regulatory light chains of a single molecule can cross-link to the light meromyosin portion of the tail. Mass spectrometric data show four specific cross-linked regions spanning residues 1428-1571 in the light meromyosin portion of the tail, consistent with cross-linking two RLC to one light meromyosin. In addition, we find that position 59 can cross-link internally to residues 42-45 within the same RLC subunit. The internal cross-link only forms in 10 S and not in unphosphorylated heavy meromyosin (lacking the light meromyosin), suggesting a structural rearrangement within the RLC attributed to the interaction of the tail with the head.
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Medicine, University of Nevada, 1664 N. Virginia Street, Reno, NV 89557, USA.
authors.forename: Bridget
authors.initials: B
authors.lastname: Salzameda
authors.forename: Kevin C
authors.initials: KC
authors.lastname: Facemyer
authors.forename: Brian W
authors.initials: BW
authors.lastname: Beck
authors.forename: Christine R
authors.initials: CR
authors.lastname: Cremo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D002850:Chromatography, Gel; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000378:metabolism*; D009218:Myosins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D009218:Myosins