CRYSTAL STRUCTURE OF A PHOSPHATASE RESISTANT MUTANT OF SPORULATION RESPONSE REGULATOR SPO0F FROM BACILLUS SUBTILIS. Madhusudan, John M. Whiteley, James A. Hoch, James Zapf, Nguyen H. Xuong* and Kottayil I. Varughese*, The Scripps Research Institute, La Jolla, CA 92037 and *University of California at San Diego, La Jolla, CA 92093-0359

Spo0F is an aspartyl pocket containing phosphotransferase in the signaling pathway controlling sporulation in Bacillus subtilis. It belongs to the superfamily of bacterial response regulatory proteins, which are activated upon phosphorylation of an invariant aspartate residue in a divalent cation dependent reaction by cognate histidine kineses. We have determined the crystal structure of a Rap phosphatase resistant mutant, Spo0F Y13S, at 1.9 Å. The structure was solved by single isomorphous replacement and anomalous I scattering techniques. The overall structural fold is ([[beta]]/[[alpha]])5 and contains a central [[beta]]-sheet. The active-site of the molecule is formed by three aspartates and a lysine at the carboxyl end of the [[beta]]-sheet and it accommodates a calcium ion. The structural analysis reveals that the overall topology and metal binding coordination at the active-site were similar to the chemotaxis response regulator CheY. Structural differences between Spo0F and CheY in the vicinity of the active-site provide insight into how similar molecular scaffolds can be adapted to perform different biological roles by alteration of a few amino acid residues. These differences may contribute to the observed stability of the phosphorylated species of Spo0F, a feature demanded by its role as a secondary messenger in the phosphorelay controlling sporulation.