Characterization of two orthologs of bacterial intramembrane metalloprotease RseP by native mass spectrometry.

Intramembrane metalloproteases are conserved in all three domains of life and regulate cellular signal transduction by cleaving membrane-anchored precursors or regulators of transcription factors. Bacterial intramembrane metalloproteases are promising targets for antimicrobial drugs as they are implicated in pathogenicity and drug resistance. Understanding the properties of intramembrane metalloproteases, particularly their interactions with zinc and inhibitors, is important for drug development. While native mass spectrometry (MS) is effective for studying intermolecular interactions, its application to membrane proteins, including intramembrane metalloproteases, presents a unique challenge for preserving noncovalent interactions in the gas phase due to their hydrophobic nature. In this study, we utilized native MS to investigate zinc and inhibitor binding to two bacterial intramembrane metalloproteases, Escherichia coli RseP (EcRseP) and its ortholog from Kangiella koreensis (KkRseP). Intact protein ions were successfully observed following optimized purification and buffer exchange protocols. Native MS revealed zinc binding to both orthologs, with EcRseP exhibiting higher affinity. In the presence of batimastat, a specific EcRseP inhibitor, both RseP orthologs formed stable complexes, demonstrating batimastat binds exclusively to zinc-bound RseP. These results demonstrate the ability of native MS for characterizing membrane protein interactions and highlight its potential as a platform for identifying specific binding events, thereby extending its established application with soluble proteins.