Dynamics of nitric oxide synthase-calmodulin interactions at physiological calcium concentrations.
The intracellular Ca²⁺ concentration is an important regulator of many cellular functions. The small acidic protein calmodulin (CaM) serves as a Ca²⁺ sensor and control element for many enzymes. Nitric oxide synthase (NOS) is one of the proteins that is activated by CaM and plays a major role in a number of key physiological and pathological processes. Previous studies have shown CaM to act like a switch that causes a conformational change in NOS to allow for the electron transfer between the reductase and oxygenase domains through a process that is thought to be highly dynamic. We have analyzed the structure and dynamics of complexes formed by peptides based on inducible NOS (iNOS) and endothelial NOS (eNOS) with CaM at Ca²⁺ concentrations that mimic the physiological basal (17 and 100 nM) and elevated levels (225 nM) found in mammalian cells using fluorescence techniques and nuclear magnetic resonance spectroscopy. The results show the CaM-NOS complexes have similar structures at physiological and fully saturated Ca²⁺ levels; however, their dynamics are remarkably different. At 225 nM Ca²⁺, the CaM-NOS complexes show overall an increase in backbone dynamics, when compared to the dynamics of the complexes at saturating Ca²⁺ concentrations. Specifically, the N-lobe of CaM in the CaM-iNOS complex displays a lower internal mobility (higher S²) and higher exchange protection compared to those of the CaM-eNOS complex. In contrast, the C-lobe of CaM in the CaM-eNOS complex is less dynamic. These results illustrate that structures of CaM-NOS complexes determined at saturated Ca²⁺ concentrations cannot provide a complete picture because the differences in intramolecular dynamics become visible only at physiological Ca²⁺ levels.
Department of Chemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
Michael
M
Piazza
Department of Chemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
J Guy
JG
Guillemette
Department of Chemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
Thorsten
T
Dieckmann
D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003619:Dansyl Compounds / Q000737:chemistry; D041961:Deuterium Exchange Measurement; D004789:Enzyme Activation; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008958:Models, Molecular; D052247:Nitric Oxide Synthase Type II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D055550:Protein Stability; D058767:Protein Unfolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D002147:Calmodulin; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D011994:Recombinant Proteins; C496319:NOS2 protein, human; C496314:NOS3 protein, human; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C029038:dansyl chloride; D002118:Calcium