PXD044750

PXD044750 is an original dataset announced via ProteomeXchange.

Title	Mapping Interactions of Calmodulin and nNOS by CXL-MS
Description	Neuronal nitric oxide synthase (nNOS) is a self-sufficient 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, greatly enhances electron transfer reactions allowing reduction of the heme and NO synthesis. Due to the dynamic nature of the nNOS reductase domain and the overall low resolution of full-length nNOS structures, the exact nature of the CaM-bound active complex during heme reduction is still unresolved. Interestingly, hydrogen-deuterium exchange and mass spectrometry (HDX-MS) studies on iNOS, but not nNOS, directly revealed interactions of the FMN-domain and CaM with the oxygenase domain. To further address this unexpected finding with nNOS, we utilized covalent crosslinking and mass spectrometry (CXL-MS) to examine interactions of CaM with full- length 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. Detailed analysis of the crosslinks provide evidence for CaM interaction with the oxygenase and reductase domain residues as well as interactions of the FMN-domain with the oxygenase dimer. Crosslink-guided docking studies reveal a conformation of nNOS that brings the FMN within 15 Å to the heme and provides further support for a more compact conformation of CaM-bound nNOS than previously observed in EM-derived structures. These studies also point to the utility of CXL-MS to capture transient dynamic conformations that may not be captured by HDX-MS experiments.
HostingRepository	PRIDE
AnnounceDate	2024-01-26
AnnouncementXML	Submission_2024-01-26_05:53:22.559.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Dana Felker
SpeciesList	scientific name: Rattus norvegicus (Rat); NCBI TaxID: 10116;
ModificationList	No PTMs are included in the dataset
Instrument	Q Exactive HF

Revision	Datetime	Status	ChangeLog Entry
0	2023-08-22 14:15:17	ID requested
⏵ 1	2024-01-26 05:53:23	announced

10.1016/j.jbc.2023.105464;

Felker D, Lee K, Pospiech TH, Morishima Y, Zhang H, Lau M, Southworth DR, Osawa Y, Mapping interactions of calmodulin and neuronal NO synthase by crosslinking and mass spectrometry. J Biol Chem, 300(1):105464(2024) [pubmed]

submitter keyword: neuronal nitric oxide synthase,CXL-MS, crosslinking

Yoichi Osawa
contact affiliation	Department of Pharmacology, University of Michigan
contact email	osawa@umich.edu
lab head
Dana Felker
contact affiliation	University of Michigan, Ann Arbor
contact email	dbfelker@umich.edu
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2024/01/PXD044750

PRIDE project URI

• PRIDE
  1. PXD044750
     1. Label: PRIDE project
     2. Name: Mapping Interactions of Calmodulin and nNOS by CXL-MS