Influence of hydrophobic mismatch on the catalytic activity of Escherichia coliGlpG rhomboid protease

Abstract

Rhomboids comprise a broad family of intramembrane serine proteases that are found in a wide range of organisms and participate in a diverse array of biological processes. Highresolution structures of the catalytic transmembrane domain of the Escherichia coli GlpG rhomboid have provided numerous insights that help explain how hydrolytic cleavage can be achieved below the membrane surface. Key to this are observations that GlpG hydrophobic domain dimensions may not be sufficient to completely span the native lipid bilayer. This formed the basis for a model where hydrophobic mismatch Induces thinning of the local membrane environment to promote access to transmembrane substrates. However, hydrophobic mismatch also has the potential to alter the functional properties of the rhomboid, a possibility we explore in the current work. For this purpose, we purified the catalytic transmembrane domain of GlpG into phosphocholine or maltoside detergent micelles of varying alkyl chain lengths, and assessed proteolytic function with a model water-soluble substrate. Catalytic turnover numbers were found to depend on detergent alkyl chain length, with saturated chains containing 10-12 carbon atoms supporting maximal activity. Similar results were obtained in phospholipid bicelles, with no proteolytic activity being detected in longer-chain lipids. Although differences in thermal stability and GlpG oligomerization could not explain these activity differences, circular dichroism spectra suggest that mismatch gives rise to a small change in structure. Overall, these results demonstrate that hydrophobic mismatch can exert an inhibitory effect on rhomboid activity, with the potential for changes in local membrane environment to regulate activity in vivo.

Interrogating Membrane Protein Conformational Dynamics within Native Lipid Compositions

“…Changes in the PE/PG ratio between C 37 and BL 37 did not seem to affect HDX significantly, whereas changes in chain length and saturation between C 37 and C 16 did. This may be related to previous evidence from detergent micelle and bicelle systems that hydrophobic mismatch could exert an inhibitory effect on GlpG activity …”

Activity‐Based Protein Profiling of Rhomboid Proteases in Liposomes

“…This indicatest hat the lipid environment influences the activity state of rhomboids, as suggested before in previousstudies. [8,24] All inhibitor screenst od ate have been performed with rhomboids solubilized in detergent micelles. Although af ew studies have used the general serine protease inhibitor 3,4-dichloroisocoumarin (DCI) to inhibit rhomboids in eukaryotic cell cultures,i ti su nclear whether the behavior of inhibitors is similar with rhomboids residing inside lipid bilayers.…”

“…[23] The ideat hatr homboid-containing micellesa re ag oodm odel system is further illustrated by two recent, separate kinetic studies thatw ere performed either in liposomeso ri nm icelles and weregenerallyi ngooda greement witheachother. [15,24] Finally,t he fluorescence microscopy performed on GUVs demonstrates that activity-based imaging inside al ipid bilayer is af easible approach. Althoughf urther development of fluo-rescentr homboid ABPs might be necessary,t his study constitutes afirst step towards fluorescencem icroscopy of rhomboid activity in membranes of living cells.…”

Micelles and Bicelles as Membrane Mimics for Membrane Protein Investigations

“…OMPLA is the first outer membrane β-barrel enzyme to be functionally characterized. 38 The E. coli pldA gene codes for a mature OMPLA protein of 269 amino acids, preceded by a signal sequence of 20 residues for protein translocation across the inner membrane. 39 The 31 kDa β-barrel protein is comprised of 12 β-strands, and its structure was determined in 1999 via X-ray crystallography.…”

“…Events such as heat shock, EDTA treatment, and phage-induced lysis negatively affect this normal lipid asymmetry. 38 These disruptions in the membrane integrity cause phospholipids that normally remain on the inner leaflet of the outer membrane to move to the outer leaflet, accessing the active site of OMPLA, triggering dimerization, and resulting in calcium-dependent phospholipase cleavage.…”