Mapping the Intersubunit Interdomain FMN-Heme Interactions in Neuronal Nitric Oxide Synthase by Targeted Quantitative Cross-Linking Mass Spectrometry

Biochemistry. 2024 Jun 4;63(11):1395-1411. doi: 10.1021/acs.biochem.4c00157. Epub 2024 May 15.

Abstract

Nitric oxide synthase (NOS) in mammals is a family of multidomain proteins in which interdomain electron transfer (IET) is controlled by domain-domain interactions. Calmodulin (CaM) binds to the canonical CaM-binding site in the linker region between the FMN and heme domains of NOS and allows tethered FMN domain motions, enabling an intersubunit FMN-heme IET in the output state for NO production. Our previous cross-linking mass spectrometric (XL MS) results demonstrated site-specific protein dynamics in the CaM-responsive regions of rat neuronal NOS (nNOS) reductase construct, a monomeric protein [Jiang et al., Biochemistry, 2023, 62, 2232-2237]. In this work, we have extended our combined approach of XL MS structural mapping and AlphaFold structural prediction to examine the homodimeric nNOS oxygenase/FMN (oxyFMN) construct, an established model of the NOS output state. We employed parallel reaction monitoring (PRM) based quantitative XL MS (qXL MS) to assess the CaM-induced changes in interdomain dynamics and interactions. Intersubunit cross-links were identified by mapping the cross-links onto top AlphaFold structural models, which was complemented by comparing their relative abundances in the cross-linked dimeric and monomeric bands. Furthermore, contrasting the CaM-free and CaM-bound nNOS samples shows that CaM enables the formation of the intersubunit FMN-heme docking complex and that CaM binding induces extensive, allosteric conformational changes across the NOS regions. Moreover, the observed cross-links sites specifically respond to changes in ionic strength. This indicates that interdomain salt bridges are responsible for stabilizing and orienting the output state for efficient FMN-heme IET. Taken together, our targeted qXL MS results have revealed that CaM and ionic strength modulate specific dynamic changes in the CaM/FMN/heme complexes, particularly in the context of intersubunit interdomain FMN-heme interactions.

MeSH terms

  • Animals
  • Binding Sites
  • Calmodulin* / chemistry
  • Calmodulin* / metabolism
  • Cross-Linking Reagents / chemistry
  • Flavin Mononucleotide* / chemistry
  • Flavin Mononucleotide* / metabolism
  • Heme* / chemistry
  • Heme* / metabolism
  • Mass Spectrometry* / methods
  • Models, Molecular
  • Nitric Oxide Synthase Type I* / chemistry
  • Nitric Oxide Synthase Type I* / metabolism
  • Protein Binding
  • Protein Domains
  • Rats

Substances

  • Nitric Oxide Synthase Type I
  • Flavin Mononucleotide
  • Heme
  • Calmodulin
  • Nos1 protein, rat
  • Cross-Linking Reagents