Allostery in recombinant soluble guanylyl cyclase from Manduca sexta

Xiaohui Hu, Lauren B. Murata, Andrzej Weichsel, Jacqueline L. Brailey, Sue A. Roberts, Alan Nighorn, William R. Montfort

Research output: Contribution to journalArticle

30 Scopus citations

Abstract

Soluble guanylyl/guanylate cyclase (sGC), the primary biological receptor for nitric oxide, is required for proper development and health in all animals. We have expressed heterodimeric full-length and N-terminal fragments of Manduca sexta sGC in Escherichia coli, the first time this has been accomplished for any sGC, and have performed the first functional analyses of an insect sGC. Manduca sGC behaves much like its mammalian counterparts, displaying a 170-fold stimulation by NO and sensitivity to compound YC-1. YC-1 reduces the NO and CO off-rates for the ∼100-kDa N-terminal heterodimeric fragment and increases the CO affinity by ∼50-fold to 1.7 μM. Binding of NO leads to a transient six-coordinate intermediate, followed by release of the proximal histidine to yield a five-coordinate nitrosyl complex (k6-5 = 12.8 s -1). The conversion rate is insensitive to nucleotides, YC-1, and changes in NO concentration up to ∼30 μM. NO release is biphasic in the absence of YC-1 (koff1 = 0.10 s-1 and koff2 = 0.0015 s-1); binding of YC-1 eliminates the fast phase but has little effect on the slower phase. Our data are consistent with a model for allosteric activation in which sGC undergoes a simple switch between two conformations, with an open or a closed heme pocket, integrating the influence of numerous effectors to give the final catalytic rate. Importantly, YC-1 binding occurs in the N-terminal two-thirds of the protein. Homology modeling and mutagenesis experiments suggest the presence of an H-NOX domain in the α subunit with importance for heme binding.

Original languageEnglish (US)
Pages (from-to)20968-20977
Number of pages10
JournalJournal of Biological Chemistry
Volume283
Issue number30
DOIs
StatePublished - Jul 25 2008

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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