Structural basis for endothelial nitric oxide synthase binding to calmodulin

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Abstract
The enzyme nitric oxide synthase (NOS) is exquisitely regulated in vivo by the Ca2+ 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 crystal lographic structure of Ca2+‐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 α‐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 Ca2+ independence of inducible NOS.