High-resolution crystal structure of Streptococcus pyogenes β-NAD⁺ glycohydrolase in complex with its endogenous inhibitor IFS reveals a highly water-rich interface

The crystal structure of the complex between the C-terminal domain of Streptococcus pyogenes β-NAD⁺ glycohydrolase and an endogenous inhibitor for SPN was determined at 1.70 Å. It reveals that the interface between the two proteins is highly rich in water molecules. One of the virulence factors produced by Streptococcus pyogenes is β-NAD⁺ glycohydrolase (SPN). S. pyogenes injects SPN into the cytosol of an infected host cell using the cytolysin-mediated translocation pathway. As SPN is toxic to bacterial cells themselves, S. pyogenes possesses the ifs gene that encodes an endogenous inhibitor for SPN (IFS). IFS is localized intracellularly and forms a complex with SPN. This intracellular complex must be dissociated during export through the cell envelope. To provide a structural basis for understanding the interactions between SPN and IFS, the complex was overexpressed between the mature SPN (residues 38–451) and the full-length IFS (residues 1–161), but it could not be crystallized. Therefore, limited proteolysis was used to isolate a crystallizable SPN_ct–IFS complex, which consists of the SPN C-terminal domain (SPN_ct; residues 193–451) and the full-length IFS. Its crystal structure has been determined by single anomalous diffraction and the model refined at 1.70 Å resolution. Interestingly, our high-resolution structure of the complex reveals that the interface between SPN_ct and IFS is highly rich in water molecules and many of the interactions are water-mediated. The wet interface may facilitate the dissociation of the complex for translocation across the cell envelope