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

Area of Science:

• Biochemistry and Molecular Biology
• Microbiology
• Structural Biology

Background:

• Bacterial intramembrane metalloproteases regulate crucial cellular processes and are key targets for novel antimicrobial drugs due to their roles in pathogenicity and resistance.
• Studying these hydrophobic membrane proteins, especially their interactions with metal ions like zinc and inhibitors, is vital for drug development.
• Native mass spectrometry (MS) is powerful for analyzing molecular interactions but faces challenges with membrane proteins due to their hydrophobic nature and the need to preserve noncovalent complexes.

Purpose of the Study:

• To investigate zinc and inhibitor binding to bacterial intramembrane metalloproteases using native MS.
• To assess the utility of native MS for characterizing interactions of hydrophobic membrane proteins.
• To explore potential drug development applications by understanding inhibitor binding specificity.

Main Methods:

• Utilized optimized purification and buffer exchange protocols to prepare bacterial intramembrane metalloprotease samples.
• Employed native mass spectrometry (MS) to analyze intact protein ions and their interactions.
• Investigated binding of zinc and the inhibitor batimastat to Escherichia coli RseP (EcRseP) and Kangiella koreensis RseP (KkRseP).

Main Results:

• Successfully observed intact protein ions for both EcRseP and KkRseP, demonstrating the feasibility of native MS for these proteins.
• Confirmed zinc binding to both RseP orthologs, with EcRseP showing a higher affinity for zinc.
• Demonstrated that the inhibitor batimastat forms stable complexes with both RseP orthologs, binding exclusively to the zinc-bound form of the proteases.

Conclusions:

• Native MS is a viable and powerful technique for characterizing interactions of bacterial intramembrane metalloproteases.
• The study provides insights into the zinc-binding properties and inhibitor interaction mechanisms of RseP orthologs.
• This work validates native MS as a platform for identifying specific binding events in membrane proteins, supporting antimicrobial drug discovery efforts.