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

Xiangdong Wu, Beatrice A. Clack, Gang Zhi, James T. Stull, Christine R. Cremo

Research output: Contribution to journalArticle

35 Scopus citations

Abstract

Smooth muscle heavy meromyosin, a double-headed proteolytic fragment of myosin lacking the COOH-terminal two-thirds of the tail, has been shown previously to be regulated by phosphorylation. To examine phosphorylation- dependent structural changes near the head-tail junction, we prepared five well regulated heavy meromyosins containing single-cysteine mutants of the human smooth muscle regulatory light chain labeled with the photocross- linking reagent, benzophenone-iodoacetamide. For those mutants that generated cross-links, only one type of cross-linked species was observed, a regulatory light chain dimer. Irradiated mutants fell into two classes. First, for Q15C, A23C, and wild type (Cys-108), a regulatory light chain dimer was formed for dephosphorylated but not thiophosphorylated heavy meromyosin. These data provide direct chemical evidence that in the dephosphorylated state, Gln-15, Ala-23, and Cys-108 on one head are positioned near (within 8.9 Å) the regulatory light chain of the partner head and that thiophosphorylation abolishes 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 thiophosphorylated states. S59C and T134C mutants were in a second mutant class, where regulatory light chain dimers were not detected in dephosphorylated or thiophosphorylated heavy meromyosin, suggesting positions outside the region of interaction of the regulatory light chains.

Original languageEnglish (US)
Pages (from-to)20328-20335
Number of pages8
JournalJournal of Biological Chemistry
Volume274
Issue number29
DOIs
StatePublished - Jul 16 1999

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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