Structural basis of substrate selectivity of E. coli prolidase

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Prolidases are conserved metalloproteases essential for cleaving Xaa-Pro dipeptides.
• Human prolidase plays a vital role in collagen recycling.
• Escherichia coli prolidase (PepQ) shares significant similarity with eukaryotic counterparts.

Purpose of the Study:

• To elucidate the structural and functional characteristics of E. coli PepQ.
• To identify key elements governing prolidase substrate specificity.
• To extend the understanding of prolidase active site mechanisms.

Main Methods:

• X-ray crystallography to determine the structure of E. coli PepQ at 2.0 Å resolution.
• Bioinformatic analysis comparing E. coli PepQ with homologous sequences and structures.
• Molecular docking simulations to predict substrate-enzyme interactions.
• Kinetic analysis using modified substrates and enzyme variants.

Main Results:

• The dimeric structure of E. coli PepQ was solved, revealing N-terminal and C-terminal domains.
• The C-terminal domain features a pita-bread fold coordinating two Mg(II) ions.
• A conserved loop with an arginine residue near the catalytic site was identified, distinct from Gram-positive bacteria and archaea.
• Docking simulations and kinetic data indicate this arginine interacts with the substrate's C-terminus.

Conclusions:

• The structure of E. coli PepQ provides insights into metalloprotease active site organization.
• A conserved arginine residue in a eukaryotic-like loop plays a critical role in substrate specificity.
• This study designates a new functional role for this key region in the prolidase active site.