title: Conformation and dynamics of the SH1-SH2 helix in scallop myosin.
abstract: Atomic structures of scallop myosin subfragment 1(S1) with the bound MgADP, MgAMPPNP, and MgADP.BeF(x) provide crystallographic evidence for a destabilization of the helix containing reactive thiols SH1 (Cys703) and SH2 (Cys693). A destabilization of this helix was not observed in previous structures of S1 (from chicken skeletal, Dictyostelium discoideum, and smooth muscle myosins), including complexes for which solution experiments indicated such a destabilization. In this study, the factors that influence the SH1-SH2 helix in scallop S1 were examined using monofunctional and bifunctional thiol reagents. The rate of monofunctional labeling of scallop S1 was increased in the presence of MgADP and MgATPgammaS but was inhibited by MgADP.V(i) and actin. The resulting changes in ATPase activities of S1 were symptomatic of SH2 and not SH1 modification, which was confirmed by mass spectrometry analysis. With bifunctional reagents of various lengths, cross-linking did not occur on a short time scale in the absence of nucleotides. In the presence of MgADP, cross-linking was greatly enhanced for all of the reagents. These reactions, as well as the formation of a disulfide bond between SH1 and SH2, were much faster in scallop S1.ADP than in rabbit skeletal S1.ADP and were rate-limited by the initial attachment of the reagent to scallop S1. The cross-linking sites were mapped by mass spectrometry to SH1 and SH2. These results reveal isoform-specific differences in the conformation and dynamics of the SH1-SH2 helix, providing a possible explanation for destabilization of this helix in some scallop S1 but not in other S1 isoform structures.
authors.affiliation: Department of Chemistry and Biochemistry and the Molecular Biology, Institute University of California, Los Angeles, California 90095, USA.
authors.forename: Lisa K
authors.initials: LK
authors.lastname: Nitao
authors.forename: Rachel R Ogorzalek
authors.initials: RR
authors.lastname: Loo
authors.forename: Elizabeth
authors.initials: E
authors.lastname: O'Neall-Hennessey
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Loo
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Szent-Györgyi
authors.forename: Emil
authors.initials: E
authors.lastname: Reisler
mesh_terms: D000818:Animals; D008974:Mollusca / Q000378:metabolism*; D009218:Myosins / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D018909:src Homology Domains
chemical_list: D009218:Myosins