1 research outputs found
Activity-Based Protein Profiling of the <i>Escherichia coli</i> GlpG Rhomboid Protein Delineates the Catalytic Core
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 <i>Escherichia coli</i> 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