Structural basis for activity regulation and substrate preference of clostridial collagenases G, H, and T

J Biol Chem. 2013 Jul 12;288(28):20184-94. doi: 10.1074/jbc.M112.448548. Epub 2013 May 23.

Abstract

Clostridial collagenases are among the most efficient enzymes to degrade by far the most predominant protein in the biosphere. Here we present crystal structures of the peptidases of three clostridial collagenase isoforms (ColG, ColH, and ColT). The comparison of unliganded and liganded structures reveals a quaternary subdomain dynamics. In the unliganded ColH structure, this globular dynamics is modulated by an aspartate switch motion that binds to the catalytic zinc. We further identified a calcium binding site in proximity to the catalytic zinc. Both ions are required for full activity, explaining why calcium critically affects the enzymatic activity of clostridial collagenases. Our studies further reveal that loops close to the active site thus serve as characteristic substrate selectivity filter. These elements explain the distinct peptidolytic and collagenolytic activities of these enzymes and provide a rational framework to engineer collagenases with customized substrate specificity as well as for inhibitor design.

Keywords: Collagenase; Metal Regulation; Protease; Protein Degradation; Protein Structure; Proteolytic Enzymes; X-ray Crystallography.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Binding Sites / genetics
  • Biocatalysis / drug effects
  • Calcium / chemistry
  • Calcium / metabolism
  • Catalytic Domain*
  • Clostridium / enzymology*
  • Clostridium / genetics
  • Clostridium histolyticum / enzymology
  • Clostridium histolyticum / genetics
  • Isoenzymes / chemistry
  • Isoenzymes / genetics
  • Isoenzymes / metabolism
  • Kinetics
  • Microbial Collagenase / chemistry*
  • Microbial Collagenase / genetics
  • Microbial Collagenase / metabolism
  • Models, Molecular*
  • Molecular Sequence Data
  • Protease Inhibitors / pharmacology
  • Protein Conformation
  • Sequence Homology, Amino Acid
  • Substrate Specificity
  • Zinc / chemistry
  • Zinc / metabolism

Substances

  • Isoenzymes
  • Protease Inhibitors
  • Microbial Collagenase
  • Zinc
  • Calcium

Associated data

  • PDB/4AQO
  • PDB/4AR1
  • PDB/4AR8
  • PDB/4AR9
  • PDB/4ARE
  • PDB/4ARF