Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 110, Issue 7, Pages 2472-2477Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1215076110
Keywords
MALDI screening; covalent inhibition; regulated intramembrane proteolysis
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Funding
- Deutsche Forschungsgemeinschaft (Emmy Noether program)
- Center for Integrated Protein Science Munich
- Marie Curie Intra-European fellowship
- MRC [MC_U105184322] Funding Source: UKRI
- Medical Research Council [MC_U105184322] Funding Source: researchfish
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Rhomboid proteases are evolutionary conserved intramembrane serine proteases. Because of their emerging role in many important biological pathways, rhomboids are potential drug targets. Unfortunately, few chemical tools are available for their study. Here, we describe a mass spectrometry-based assay to measure rhomboid substrate cleavage and inhibition. We have identified isocoumarin inhibitors and developed activity-based probes for rhomboid proteases. The probes can distinguish between active and inactive rhomboids due to covalent, reversible binding of the active-site serine and stable modification of a histidine residue. Finally, the structure of an isocoumarin-based inhibitor with Escherichia coli rhomboid GlpG uncovers an unusual mode of binding at the active site and suggests that the interactions between the 3-substituent on the isocoumarin inhibitor and hydrophobic residues on the protease reflect S' subsite binding. Overall, these probes represent valuable tools for rhomboid study, and the structural insights may facilitate future inhibitor design.
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