Mapping the H-NOX/HK Binding Interface in Vibrio cholerae by Hydrogen/Deuterium Exchange Mass Spectrometry

Heme-nitric oxide/oxygen binding (H-NOX) proteins are a group of hemoproteins that bind diatomic gas ligands such as nitric oxide (NO) and oxygen (O₂). H-NOX proteins typically regulate histidine kinases (HK) located within the same operon. It has been reported that NO-bound H-NOXs inhibit cognate histidine kinase autophosphorylation in bacterial H-NOX/HK complexes; however, a detailed mechanism of NO-mediated regulation of the H-NOX/HK activity remains unknown. In this study, the binding interface of Vibrio cholerae ( Vc) H-NOX/HK complex was characterized by hydrogen/deuterium exchange mass spectrometry (HDX-MS) and further validated by mutagenesis, leading to a new model for NO-dependent kinase inhibition. A conformational change in Vc H-NOX introduced by NO generates a new kinase-binding interface, thus locking the kinase in an inhibitory conformation.