The formation of a complex between calmodulin and neuronal nitric oxide synthase is determined by ESI-MS.
Calmodulin (CaM) is an acidic ubiquitous calcium binding protein, involved in many intracellular processes, which often involve the formation of complexes with a variety of protein and peptide targets. One such system, activated by Ca2+ loaded CaM, is regulation of the nitric oxide synthase (NOS) enzymes, which in turn control the production of the signalling molecule and cytotoxin NO. A recent crystallographic study mapped the interaction of CaM with endothelial NOS (eNOS) using a 20 residue peptide comprising the binding site within eNOS. Here the interaction of CaM to the FMN domain of neuronal nitric oxide synthase (nNOS) has been investigated using electrospray ionization mass spectrometry (ESI-MS). The 46 kDa complex formed by CaM-nNOS has been retained in the gas-phase, and is shown to be exclusively selective for CaM.4Ca2+. Further characterization of this important biological system has been afforded by examining a complex of CaM with a 22 residue synthetic peptide, which represents the linker region between the reductase and oxygenase domains of nNOS. This nNOS linker peptide, which is found to be random coil in aqueous solution by both circular dichroism and molecular modelling, also exhibits great discrimination for the form of CaM loaded with 4[Ca2+]. The peptide binding loop is presumed to be configured to an alpha-helix on binding to CaM as was found for the related eNOS binding peptide. Our postulate is supported by gas-phase molecular dynamics calculations performed on the isolated nNOS peptide. Collision induced dissociation was employed to probe the strength of binding of the nNOS binding peptide to CaM.4Ca2+. The methodology taken here is a new approach in understanding the CaM-nNOS binding site, which could be employed in future to inform the specificity of CaM binding to other NOS enzymes.
Shirran
Sally
S
University of Edinburgh, School of Chemistry, West Mains Road, Edinburgh EH9 3JJ, UK.
Garnaud
Pierre
P
Daff
Simon
S
McMillan
Derek
D
Barran
Perdita
P
D001665:Binding Sites; D002147:Calmodulin / Q000737:chemistry* / Q000648:ultrastructure*; D003198:Computer Simulation; D004789:Enzyme Activation; D008956:Models, Chemical; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry / Q000648:ultrastructure; D052248:Nitric Oxide Synthase Type I / Q000737:chemistry* / Q000648:ultrastructure*; D011485:Protein Binding; D011487:Protein Conformation; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D002147:Calmodulin; D046912:Multiprotein Complexes; D052248:Nitric Oxide Synthase Type I