Mapping Interactions of Calmodulin and nNOS by CXL-MS

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.