Structural basis for specific calcium binding by the polycystic-kidney-disease domain of Vibrio anguillarum protease Epp

Highlights: • Crystal structure of the first PKD domain of Vibrio anguillarum Epp is determined. • Epp-PKD1 contains a pentagonal-bipyramidal Ca²⁺-binding site. • Specific binding to Ca²⁺ increases conformational stability of Epp-PKD1. • Epp-PKD1 differs in the oligomeric state and Ca²⁺ coordination from the homologous PrtV-PKD1. Extracellular proteases are often produced as pre-pro-enzyme and then undergo multiple processing steps to mature into the active form. The protease Epp, a virulent factor of Vibrio anguillarum, belongs to this family. Its maturation might be regulated by Ca²⁺ via its polycystic kidney disease (PKD) domain, but the molecular mechanism is unknown. Herein, we report the crystal structure of the first PKD domain from V. anguillarum Epp (Epp-PKD1) and its specific Ca²⁺-binding capacity. Epp-PKD1 exists as a monomer, consisting of seven β-strands which form two β-sheets stacking with each other. One Ca²⁺ is bound by the residues Asn3, Gln4, Asp27, Asp29, Asp68 and a water molecule with a pentagonal bipyramidal geometry. Incubating the apo Epp-PKD1 with Ca²⁺ but not Mg²⁺, Mn²⁺, or Zn²⁺, enhances the thermal and chemical stability of Epp-PKD1, indicating its specific binding to Ca²⁺. Epp-PKD1 shares high similarity in both sequence and overall structure with that of Vibrio cholerae PrtV, a homologous protease of Epp, however, they differ in the oligomeric state and local structure at the Ca²⁺-binding site, suggesting maturation of PrtV and Epp might be differently regulated by Ca²⁺. Likely, proteases may take advantage of the structural diversity in PKD domains to tune their Ca²⁺-regulated maturation process.