Structural basis for endothelial nitric oxide synthase binding to calmodulin

Abstract

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-speci®c differences. The a-helical eNOS peptide binds in an antiparallel orientation to CaM through extensive hydrophobic interactions. Unique NOS interactions occur with: (i) the CaM¯exible central linker, explaining its importance in NOS activation; and (ii) the CaM C-terminus, explaining the NOS-speci®c 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 speci®c hydrophobic residues in the Ca 2+ independence of inducible NOS.

Thermodynamics of apocalmodulin and nitric oxide synthase II peptide interaction

“…This is consistent with the fact that position 13 varies among all NOS isoforms [15] and therefore does not require a hydrophobic amino acid.…”

“…Aoyagi et al [15] proposed in a recent study that hydrophobic residues V517 and L523 (human NOS-II) might represent additional important hydrophobic anchors for interaction with ApoCaM. The corresponding amino acids in our study are V511 and F517 (mouse NOS-II).…”

Calmodulin phosphorylation and modulation of endothelial nitric oxide synthase catalysis

“…Although eNOS and nNOS have similar features, their amino acid sequences are only 60% identical. Important differences exist in terms of the biological function and catalysis of these NOS isoforms and in the nature of their interaction with CaM (28)(29)(30)(31)(32)(33). Given these disparities, it is perhaps not surprising that the phosphorylation of CaM affects the kinetics of eNOS and nNOS differently.…”

Developing master keys to brain pathology, cancer and aging from the structural biology of proteins controlling reactive oxygen species and DNA repair

“…However, the structure and biochemical analysis of the nNOSred dimer provided a template for a model of the holo-nNOS enzyme assembly (Garcin et al, 2004). The NOS model was built by connecting the dimeric NOSox modules and a CaM: NOS-peptide complex (Aoyagi et al, 2003) to the NOSred structure. Model building was constrained by the short CaM binding linker between NOSox to NOSred.…”

“…Moreover, the pilus of Neisseria gonorrhoeae, which shares substantial sequence conservation with N. meningitides, acts cooperatively with a porin to induce calcium ion transients in infected epithelial cells (Ayala et al, 2005b). Such calcium mediated signaling by pathogen invasion in neuronal cells is expected to increase ROS stress (Koedel and Pfister, 1999), through the calcium-mediated activation of neuronal NOS (Aoyagi et al, 2003). Therefore, a combination of factors may exacerbate ROS production and provide increased risk of damage to the brain.…”