Activity-based probes for rhomboid proteases discovered in a mass spectrometry-based assay.
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.
Center for Integrated Protein Science Munich, Lehrstuhl für Chemie der Biopolymere, Technische Universität München, 85354 Freising, Germany.
Oliver
O
Vosyka
Kutti R
KR
Vinothkumar
Eliane V
EV
Wolf
Arwin J
AJ
Brouwer
Rob M J
RM
Liskamp
Steven H L
SH
Verhelst
D057930:Click Chemistry; D003460:Crystallization; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D010450:Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D049934:Isocoumarins / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D015335:Molecular Probes / Q000737:chemistry* / Q000378:metabolism; D015394:Molecular Structure; D059748:Proteolysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013379:Substrate Specificity
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C102898:GlpG protein, E coli; D049934:Isocoumarins; D008565:Membrane Proteins; D015335:Molecular Probes; D010450:Endopeptidases