W0060

Structural Basis for Phosphate Transfer in the Bacterial Phosphorylation Cascade. Piotr Sliz¹, Annie Cunningham¹, Brigitte Koch², Wolfgang Hengstenberg², Emil F. Pai¹, ¹Department of Biochemistry, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada, ²Fakultat Biologie, Arbeitsgruppe Physiologie der Mikroorganismen, Ruhr-Universitat, Bochum, D-44780, Germany

We have determined the structure of Asp81Leu mutant of the L.lactis enzyme IIA (EIIA) from the lactose specific phosphoenolpyruvate system (PTS). The structure of the wild type EIIA, homotrimeric protein forming a nine helical bundle was reported by us last year. In the wild-type protein, in addition to hydrophobic interactions, the subunits of EIIA are stabilized by a centrally located divalent metal ion. The function of the metal was unclear and we had postulated that it may play a structural or regulatory function. It was surprising to find that the mutation of residues coordinating the metal ion, Asp81 to leucine resulted in a protein which still possesses some functionality.

The crystallization conditions for the mutant protein are different from the wild type EIIA, but the protein still crystallized in the same space group. The complete data set using frozen crystals was collected using a rotating anode source and structure has been solved to 3.1Å. The fold of the protein is identical to the wild type EIIA, suggesting that the metal ion is not required for the structural stability of the protein. The only differences between the two forms of EIIA are in the region of the active site histidines. The orientation of His78, which normally is phosphorylated by HPr and then transfers the phosphate to another PTS protein, enzyme IIB has changed. The residue does not interact with water molecules, which in wild type EIIA were stabilized by the bonding network originating from the metal ion. The lack of interaction makes the residue more flexible. The biochemical data suggest that even though the mutant of EIIA can still become phosphorylated by HPr, the protein cannot transfer the phosphate to enzyme IIB. In light of this new structural information, we postulate that this is due to the improper orientation of His78 which is lacking the required stability.