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

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Intramembrane protease mechanisms remain poorly understood due to limited structural data of substrate complexes.
• Rhomboid proteases are a key family of intramembrane proteases with incompletely elucidated substrate interactions.

Purpose of the Study:

• To investigate substrate binding by rhomboid proteases, specifically Escherichia coli GlpG.
• To gain structural and enzymological insights into the interactions between GlpG and its substrates.

Main Methods:

• Synthesis of novel peptidyl-chloromethylketone (CMK) inhibitors derived from the TatA substrate.
• Enzymological analysis and co-crystallography of GlpG with peptidyl-CMK inhibitors.
• Molecular dynamics simulations to model the Michaelis complex.

Main Results:

• Peptidyl-CMKs bind GlpG in a substrate-like manner, revealing S1-S4 subsites.
• The S1 subsite merges with the 'water retention site', indicating interplay between binding and catalysis.
• The S4 subsite is plastic, formed by the L1 loop, suggesting a conserved binding function in rhomboid-like proteins.

Conclusions:

• Structural and enzymological data provide a detailed understanding of substrate binding in rhomboid proteases.
• The plasticity of the S4 subsite and the L1 loop's role in substrate recognition are key findings.
• The study proposes a conserved substrate/client-protein binding function for homologous regions in the rhomboid-like superfamily.