5F5D

Crystal structures and Inhibition kinetics reveal a two-state catalytic mechanism with drug design implications for rhomboid proteolysis

Summary for 5F5D

• Entry DOI: 10.2210/pdb5f5d/pdb
• Related: 5F5B 5F5G 5F5J 5F5K
• Descriptor: Rhomboid protease GlpG (2 entities in total)
• Functional Keywords: intramembrane protease, rhomboid, bicelle, hydrolase
• Biological source: Escherichia coli
• Cellular location: Cell inner membrane ; Multi-pass membrane protein . Cell membrane ; Multi-pass membrane protein : A0A0J2E248
• Total number of polymer chains: 1
• Total formula weight: 23800.13

Authors

Urban, S.,Cho, S.,Dickey, S.W. (deposition date: 2015-12-04, release date: 2016-02-24, Last modification date: 2023-09-27)

Primary citation

Cho, S.,Dickey, S.W.,Urban, S.
Crystal Structures and Inhibition Kinetics Reveal a Two-Stage Catalytic Mechanism with Drug Design Implications for Rhomboid Proteolysis.
Mol.Cell, 61:329-340, 2016

PubMed 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.

PubMed: 26805573
DOI: 10.1016/j.molcel.2015.12.022

Experimental method

X-RAY DIFFRACTION (2.5 Å)