Structural Studies of Phosphatidylinositol-Specific Phospholipase C from S. Aureus; An Intramolecular π-Cation Latch

Abstract

Staphylococcus aureus secretes a phosphatidylinositol-specific phospholipase C (PIPLC) that contributes to bacterial virulence. We have determined the crystal structure of this enzyme at pH 4.6 and pH 7.5. When crystallized under slightly basic conditions (pH 7.5), the S. aureus PIPLC (SaPI-PLC) structure closely follows the conformation of other PI-PLCs. However, when crystallized under acidic conditions (pH 4.6), a large section of mobile loop at the αβ-barrel rim in the vicinity of the active site shows ∼10 A shift from the position of this loop in all other published PI-PLC structures. The cause of this loop shift under acidic conditions is the result of a titratable intramolecular π-cation interaction between His258 and Phe249. A structure of the mutant protein H258Y crystallized at pH 4.6 does not exhibit this interaction, and the mobile loop remains in the open position. The π-cation latched mobile loop, when in the closed position, can restrict substrate access to the active site. The pH profile for enzyme activity exhibits a maximum at pH 6.5 with steep decreases at pH 6 and 7. Studies of the enzyme binding to vesicles indicate the Kd is lower at acidic pH values (consistent with electrostatic interactions dominating binding) and increases dramatically above pH 7. Thus, rather than affect bulk binding of the protein to target vesicles, this titratable π-histidine cation interaction must affect access of the substrate in the active site.