Structural basis for endothelial nitric oxide synthase binding to calmodulin.
The enzyme nitric oxide synthase (NOS) is exquisitely regulated in vivo by the Ca(2+) sensor protein calmodulin (CaM) to control production of NO, a key signaling molecule and cytotoxin. The differential activation of NOS isozymes by CaM has remained enigmatic, despite extensive research. Here, the crystallographic structure of Ca(2+)-loaded CaM bound to a 20 residue peptide comprising the endothelial NOS (eNOS) CaM-binding region establishes their individual conformations and intermolecular interactions, and suggests the basis for isozyme-specific differences. The alpha-helical eNOS peptide binds in an antiparallel orientation to CaM through extensive hydrophobic interactions. Unique NOS interactions occur with: (i). the CaM flexible central linker, explaining its importance in NOS activation; and (ii). the CaM C-terminus, explaining the NOS-specific requirement for a bulky, hydrophobic residue at position 144. This binding mode expands mechanisms for CaM-mediated activation, explains eNOS deactivation by Thr495 phosphorylation, and implicates specific hydrophobic residues in the Ca(2+) independence of inducible NOS.
Aoyagi
Mika
M
Department of Molecular Biology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Arvai
Andrew S
AS
Tainer
John A
JA
Getzoff
Elizabeth D
ED
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004727:Endothelium / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D007527:Isoenzymes / Q000378:metabolism; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats
D002147:Calmodulin; D007527:Isoenzymes; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D002118:Calcium