Substrate binding and specificity of rhomboid intramembrane protease revealed by substrate-peptide complex structures

The mechanisms of intramembrane proteases are incompletely understood due to the lack of structural data on substrate complexes. To gain insight into substrate binding by rhomboid proteases, we have synthesised a series of novel peptidyl‐chloromethylketone (CMK) inhibitors and analysed their interactions with Escherichia coli rhomboid GlpG enzymologically and structurally. We show that peptidyl‐CMKs derived from the natural rhomboid substrate TatA from bacterium Providencia stuartii bind GlpG in a substrate‐like manner, and their co‐crystal structures with GlpG reveal the S1 to S4 subsites of the protease. The S1 subsite is prominent and merges into the ‘water retention site’, suggesting intimate interplay between substrate binding, specificity and catalysis. Unexpectedly, the S4 subsite is plastically formed by residues of the L1 loop, an important but hitherto enigmatic feature of the rhomboid fold. We propose that the homologous region of members of the wider rhomboid‐like protein superfamily may have similar substrate or client‐protein binding function. Finally, using molecular dynamics, we generate a model of the Michaelis complex of the substrate bound in the active site of GlpG. Synopsis Despite valuable insights from inhibitor‐bound rhomboid proteases, full understanding of intramembrane proteolysis requires structural views of protease‐substrate complexes. Direct insights into substrate binding and catalytic mechanism of an intramembrane protease now come from X‐ray structures of the bacterial rhomboid protease GlpG complexed to substrate‐derived peptide inhibitors. Peptidyl chloromethylketones are substrate‐mimicking, mechanism‐based inhibitors of rhomboid protease. Structures of inhibitor‐bound complexes reveal the S1 to S4 subsites and explain GlpG substrate specificity. The S1 subsite is juxtaposed to the proposed ‘water retention site’. The conserved GlpG L1 loop forms the S4 subsite, a structural principle possibly common to other members of the rhomboid‐like superfamily. Structure‐based modeling and molecular dynamics simulations allow generating a model of the Michaelis complex with the substrate. Graphical Abstract X‐ray structures of rhomboid protease GlpG complexed to substrate‐derived peptide inhibitors provide direct insights into substrate binding and mechanism of an intramembrane protease.