Nitric oxide modification of rat brain neurogranin. Identification of the cysteine residues involved in intramolecular disulfide bridge formation using site-directed mutagenesis.
Neurogranin (Ng) is a neuron-specific protein kinase C-selective substrate, which binds calmodulin (CaM) in the dephosphorylated form at low levels of Ca2+. This protein contains redox active Cys residues that are readily oxidized by several nitric oxide (NO) donors and other oxidants to form intramolecular disulfide. Identification of the Cys residues of rat brain Ng, Cys3, Cys4, Cys9, and Cys51, involved in NO-mediated intramolecular disulfide bridge formation was examined by site-directed mutagenesis. Mutation of all four Cys residues or single mutation of Cys51 blocked the oxidant-mediated intramolecular disulfide formation as monitored by the downward mobility shift under nonreducing SDS-polyacrylamide gel electrophoresis. Single mutation of Cys3, Cys4, or Cys9 or double mutation of any pair of these three Cys residues did not block such intramolecular disulfide formation, although the rates of oxidation of these mutant proteins were different. Thus, Cys51 is an essential pairing partner in NO-mediated intramolecular disulfide formation in Ng. Cys3, Cys4, and Cys9 individually could pair with Cys51, and the order of reactivity was Cys9 > Cys4 > Cys3, suggesting that Cys9 and Cys51 form the preferential disulfide bridge. In all cases tested, the intramolecularly disulfide bridged Ng proteins displayed dramatically attenuated CaM-binding affinity and approximately 2-3-fold weaker protein kinase C substrate phosphorylation activity. The data indicate that the N-terminal Cys3, Cys4, and Cys9 are in close proximity to the C-terminal Cys51 in solution. The disulfide bridge between the N- and C-terminal domains of Ng renders the central CaM-binding and phosphorylation site domain in a fixed conformation unfavorable for binding to CaM and as a substrate of protein kinase C.
Section on Metabolic Regulation, Endocrinology and Reproduction Research Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892-4510, USA.
C W
CW
Mahoney
J H
JH
Pak
K P
KP
Huang
D000818:Animals; D002148:Calmodulin-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D002460:Cell Line; D003545:Cysteine / Q000032:analysis*; D004220:Disulfides / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics; D051579:Neurogranin; D009569:Nitric Oxide / Q000737:chemistry*; D051381:Rats
D002148:Calmodulin-Binding Proteins; D004220:Disulfides; D009419:Nerve Tissue Proteins; C496651:Nrgn protein, rat; D051579:Neurogranin; D009569:Nitric Oxide; D003545:Cysteine