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

EMBO J. 2014 Oct 16;33(20):2408-21. doi: 10.15252/embj.201489367. Epub 2014 Sep 12.


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.

Keywords: intramembrane protease; rhomboid family; rhomboid protease; structure; substrate recognition.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Chloromethyl Ketones / chemical synthesis
  • Amino Acid Chloromethyl Ketones / pharmacology*
  • Binding Sites
  • Catalytic Domain
  • Crystallography, X-Ray
  • DNA-Binding Proteins / antagonists & inhibitors
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Endopeptidases / chemistry*
  • Endopeptidases / genetics
  • Endopeptidases / metabolism
  • Escherichia coli / chemistry
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics
  • Escherichia coli Proteins / antagonists & inhibitors
  • Escherichia coli Proteins / chemistry*
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • Membrane Proteins / antagonists & inhibitors
  • Membrane Proteins / chemistry*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Models, Molecular*
  • Molecular Dynamics Simulation*
  • Mutation
  • Protein Binding
  • Providencia / chemistry*
  • Recombinant Proteins
  • Substrate Specificity


  • Amino Acid Chloromethyl Ketones
  • DNA-Binding Proteins
  • Escherichia coli Proteins
  • GlpG protein, E coli
  • Membrane Proteins
  • Recombinant Proteins
  • Endopeptidases

Associated data

  • PDB/4QNZ
  • PDB/4QO0
  • PDB/4QO2