Crystal Structures and Inhibition Kinetics Reveal a Two-Stage Catalytic Mechanism with Drug Design Implications for Rhomboid Proteolysis

Mol Cell. 2016 Feb 4;61(3):329-340. doi: 10.1016/j.molcel.2015.12.022. Epub 2016 Jan 21.

Abstract

Intramembrane proteases signal by releasing proteins from the membrane, but despite their importance, their enzymatic mechanisms remain obscure. We probed rhomboid proteases with reversible, mechanism-based inhibitors that allow precise kinetic analysis and faithfully mimic the transition state structurally. Unexpectedly, inhibition by peptide aldehydes is non-competitive, revealing that in the Michaelis complex, substrate does not contact the catalytic center. Structural analysis in a membrane revealed that all extracellular loops of rhomboid make stabilizing interactions with substrate, but mainly through backbone interactions, explaining rhomboid's broad sequence selectivity. At the catalytic site, the tetrahedral intermediate lies covalently attached to the catalytic serine alone, with the oxyanion stabilized by unusual tripartite interactions with the side chains of H150, N154, and the backbone of S201. We also visualized unexpected substrate-enzyme interactions at the non-essential P2/P3 residues. These "extra" interactions foster potent rhomboid inhibition in living cells, thereby opening avenues for rational design of selective rhomboid inhibitors.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Aldehydes / chemistry
  • Aldehydes / metabolism
  • Aldehydes / pharmacology*
  • Anti-Bacterial Agents / chemistry
  • Anti-Bacterial Agents / metabolism
  • Anti-Bacterial Agents / pharmacology*
  • Catalysis
  • Crystallography, X-Ray*
  • DNA-Binding Proteins / antagonists & inhibitors*
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Drug Design*
  • Endopeptidases / chemistry
  • Endopeptidases / genetics
  • Endopeptidases / metabolism
  • Enzyme Stability
  • Escherichia coli Proteins / antagonists & inhibitors*
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • Kinetics
  • Membrane Proteins / antagonists & inhibitors*
  • Membrane Proteins / chemistry
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Models, Molecular
  • Molecular Targeted Therapy*
  • Peptides / chemistry
  • Peptides / metabolism
  • Peptides / pharmacology*
  • Protease Inhibitors / chemistry
  • Protease Inhibitors / metabolism
  • Protease Inhibitors / pharmacology*
  • Protein Binding
  • Protein Conformation
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Structure-Activity Relationship
  • Substrate Specificity

Substances

  • Aldehydes
  • Anti-Bacterial Agents
  • DNA-Binding Proteins
  • Escherichia coli Proteins
  • GlpG protein, E coli
  • Membrane Proteins
  • Peptides
  • Protease Inhibitors
  • Recombinant Proteins
  • Endopeptidases