8t1k | pdb_00008t1k
DSBU crosslinked nNOS-CaM oxygenase homodimer
Structural highlights
FunctionNOS1_RAT Produces nitric oxide (NO) which is a messenger molecule with diverse functions throughout the body. In the brain and peripheral nervous system, NO displays many properties of a neurotransmitter. Inhibitory transmitter for non-adrenergic and non-cholinergic nerves in the colorectum. Probably has nitrosylase activity and mediates cysteine S-nitrosylation of cytoplasmic target proteins such SRR. Inhibitory transmitter for non-adrenergic and non-cholinergic nerves in the colorectum. Publication Abstract from PubMedNeuronal nitric oxide synthase (nNOS) is a homodimeric cytochrome P450-like enzyme that catalyzes the conversion of L-arginine to nitric oxide in the presence of NADPH and molecular oxygen. The binding of calmodulin (CaM) to a linker region between the FAD/FMN containing reductase domain and the heme containing oxygenase domain is needed for electron transfer reactions, reduction of the heme, and NO synthesis. Due to the dynamic nature of the reductase domain and low resolution of available full-length structures, the exact conformation of the CaM-bound active complex during heme reduction is still unresolved. Interestingly, hydrogen-deuterium exchange and mass spectrometry (HDX-MS) studies revealed interactions of the FMN-domain and CaM with the oxygenase domain for iNOS, but not nNOS. This finding prompted us to utilize covalent crosslinking and mass spectrometry (CXL-MS) to clarify interactions of CaM with nNOS. Specifically, MS-cleavable bifunctional crosslinker disuccinimidyl dibutyric urea was used to identify thirteen unique crosslinks between CaM and nNOS as well as 61 crosslinks within the nNOS. The crosslinks provided evidence for CaM interaction with the oxygenase and reductase domain residues as well as interactions of the FMN-domain with the oxygenase dimer. Cryo-EM studies, which gave a high-resolution model of the oxygenase domain, along with crosslink-guided docking provided a model of nNOS that brings the FMN within 15 A of the heme in support for a more compact conformation than previously observed. These studies also point to the utility of CXL-MS in capturing transient dynamic conformations that may not be captured by HDX-MS experiments. Mapping interactions of calmodulin and neuronal NO synthase by crosslinking and mass spectrometry.,Felker D, Lee K, Pospiech TH, Morishima Y, Zhang H, Lau M, Southworth D, Osawa Y J Biol Chem. 2023 Nov 16:105464. doi: 10.1016/j.jbc.2023.105464. PMID:37979917[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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