Background: Bacterial collagenases degrade collagen substrates with high efficiency yet varying specificity.
Results: The newly identified calcium site, aspartate switch, and conformational selectivity filter regulate substrate access to the active sites of these collagenases.
Conclusion: The unanticipated dynamics of the substrate recognition sites plus zinc occupancy combine to tune the enzymatic activity.
Significance: The crystal structures provide a rational framework to understand and optimize the isoform-dependent collagenase activities.
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.