Activity-Based Protein Profiling of the Escherichia coli GlpG Rhomboid Protein Delineates the Catalytic Core

Abstract

Rhomboid proteins comprise the largest class of intramembrane protease known, being conserved from bacteria to humans. The functional status of these proteases is typically assessed through direct or indirect detection of peptide cleavage products. Although these assays can report on the ability of a rhomboid to catalyze peptide bond cleavage, differences in measured hydrolysis rates can reflect changes in the structure and activity of catalytic residues, as well as the ability of the substrate to access the active site. Here we show that a highly reactive and sterically unencumbered fluorophosphonate activity-based protein profiling probe can be used to report on the catalytic integrity of active site residues in the Escherichia coli GlpG protein. We used results obtained with this probe on GlpG in proteomic samples, in combination with a conventional assay of proteolytic function on purified samples, to identify residues that are located on the cytoplasmic side of the lipid bilayer that are required for maximal proteolytic activity. Regions tested include the 90-residue aqueous-exposed N-terminus that encompasses a globular structure that we have determined by solution nuclear magnetic resonance, along with residues on the cytoplasmic side of the transmembrane domain core. While in most cases mutation or elimination of these residues did not significantly alter the catalytic status of the GlpG active site, the lipid-facing residue Arg227 was found to be important for maintaining a catalytically competent active site. In addition, we found a functionally critical region outside the transmembrane domain (TMD) core that is required for maximal protease activity. This region encompasses an additional 8-10 residues on the N-terminal side of the TMD core that precedes the first transmembrane segment and was not previously known to play a role in rhomboid function. These findings highlight the utility of the activity-based protein profiling approach for the characterization of rhomboid function.

Activity-based probes for rhomboid proteases discovered in a mass spectrometry-based assay

“…One other isocoumarin (JLK-6; 20, Table S1) has been reported to inhibit E. coli rhomboid GlpG (23). Sulfonylated β-lactams recently were found to inhibit bacterial rhomboids (24), as well as two fluorophosphonates (25,26).In this work, we present a unique rhomboid inhibition assay that monitors the cleavage of a protein substrate by MALDI mass spectrometry (MS). In a screen of small molecules, we discovered inhibitors and ABPs for bacterial rhomboids.…”

“…One other isocoumarin (JLK-6; 20, Table S1) has been reported to inhibit E. coli rhomboid GlpG (23). Sulfonylated β-lactams recently were found to inhibit bacterial rhomboids (24), as well as two fluorophosphonates (25,26).…”

Inhibitors of rhomboid proteases

“…Since they only react with the active enzyme species, they can be utilized as a read-out of an enzyme's activity state. For rhomboids, several activity-based probes have been reported [35,37,45]. They enable a variety of different detection methods including gel-based analysis [35,37,45], fluorescent polarization in 96-well plate format [35], and fluorescent microscopy [46].…”

“…They are based on the broad spectrum serine protease and esterase inhibitor DFP. Although DFP only acts as a weak rhomboid protease inhibitor, the fluorophosphonate ABPs react with the active site serine of GlpG at concentrations as low as 10 nM [45]. More importantly, these ABPs react with rhomboid proteases from a wide variety of organisms (Wolf & Verhelst, unpublished observation).…”

“…4A) to identify residues in GlpG that are directly involved in the formation of an intact catalytic site [45]. Wolf et al developed an inhibitor screening method for GlpG based on fluorescent polarization with a FP-Rh [35], a technique originally developed as a high-throughput screening method for poorly characterized soluble enzymes without the necessity of having a substrate [60].…”