Regulation of mammalian nitric oxide synthases by electrostatic interactions in the linker region of calmodulin.
Calmodulin (CaM), the ubiquitous Ca(2+)-sensing protein, consists of two globular domains separated by a flexible central linker that properly orients CaM's globular domains to bind and regulate various intracellular proteins, including the nitric oxide synthase (NOS) enzymes. In the present study we determined that the charge and length of the central linker of CaM has an effect on the binding and activation of the NOS isozymes by using a variety of charge CaM mutants (T79D, S81D, T79D/S81D, S101D and E84R/E87K) and CaM mutants with residues removed (Delta84, Delta83-84, and Delta81-84). Our kinetic and spectropolarimetry results demonstrate that the NOS enzymes are not adversely affected by the CaM mutants with the exceptions of S101D, E84R/E87K and the deletion of residue 84. Electrostatic interactions in the central linker between residues 82-87 in combination with hydrophobic interactions in the globular domains of CaM are important for its tight association to inducible NOS.
Spratt
Donald E
DE
Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1.
Israel
Odisho K
OK
Taiakina
Valentina
V
Guillemette
J Guy
JG
D019943:Amino Acid Substitution; D000818:Animals; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D057927:Hydrophobic and Hydrophilic Interactions; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D052247:Nitric Oxide Synthase Type II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020836:Protein Structure, Quaternary / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D051381:Rats; D055672:Static Electricity
D002147:Calmodulin; D007527:Isoenzymes; D046912:Multiprotein Complexes; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat