8T1J

Uncrosslinked nNOS-CaM oxygenase homodimer

8T1J の概要

• エントリーDOI: 10.2210/pdb8t1j/pdb
• EMDBエントリー: 40969
• 分子名称: Nitric oxide synthase 1, ZINC ION, ARGININE, ... (5 entities in total)
• 機能のキーワード: calmodulin, complex, cytosolic protein
• 由来する生物種: Rattus norvegicus (Norway rat)
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 323670.42

構造登録者

Lee, K.,Pospiech, T.H.,Southworth, D. (登録日: 2023-06-02, 公開日: 2023-11-29, 最終更新日: 2023-12-27)

主引用文献

Felker, D.,Lee, K.,Pospiech, T.H.,Morishima, Y.,Zhang, H.,Lau, M.,Southworth, D.R.,Osawa, Y.
Mapping interactions of calmodulin and neuronal NO synthase by crosslinking and mass spectrometry.
J.Biol.Chem., 300:105464-105464, 2023

PubMed Abstract: Neuronal 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 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 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 Å of the heme in support for a more compact conformation than previously observed. These studies also point to the utility of covalent crosslinking and mass spectrometry in capturing transient dynamic conformations that may not be captured by hydrogen-deuterium exchange and mass spectrometry experiments.

PubMed: 37979917
DOI: 10.1016/j.jbc.2023.105464

実験手法

ELECTRON MICROSCOPY (2.7 Å)