The objective of this proposal is to use X-ray crystallographic techniques to study the structure and function of the regulatory proteins involved in the sporulation of Bacillus subtilis. The main components of the regulatory switch for sporulation are the ATP dependent kinases KinA and KinB, and the response regulatory proteins - Spo0F, Spo0B and Spo0A. The phosphorelay is initiated when the kinases phosphorylate the second messenger Spo0F. Spo0F-P is the substrate for Spo0B, a phosphoprotein phosphotransferase which transfers its phosphate to a key Asp residue in Spo0A. When conditions for growth deteriorate, the cellular levels of Spo0A-P increase and the abB gene becomes repressed and transcription of the sporulation genes are initiated. Spo0A is the sole transcription factor in this complex cellular process of sporulation. The proposal will focus on the structure and properties of Spo0F, Spo0B and Spo0A. The key questions to be resolved are (i) What makes the phosphate more stable in Spo0F than in the analogous protein of chemotaxis, CheY? (ii) What is the mechanism of phosphate transfer to and from Spo0F? Site directed mutations will be employed to identify residues important in the interactions with KinA, Spo0B and phophatases, (iii) Which His residue gets phosphorylated in Spo0B and what is the environment of this residue? (iv) How does the phosphorylation of the N-terminal domain of Spo0A influence the DNA binding properties of its C-terminal domain? The crystal structure of Spo0F has been determined. In addition, the structure of a Y13S mutant that is resistant to the phosphatase activity of Spo0L has also been determined. Spo0B has been crystallized. Diffraction data on the native crystal to 2.9 Angstrom, a gold derivative to 3.0 Angstrom, a Pt derivative to 3.2 Angstrom and Hg derivative to 3.8 Angstrom have been measured already. Epo0A is a two domain protein. The N-terminal domain is homologous to Spo0F and also contains an aspartic acid that can be phosphorylated. The C-terminal domain contains the DNA interacting portions. The N and C-terminal domains of Spo0A and the whole protein have been overexpressed in E. coli and purified. Crystallization is in progress as the first step towards structural characterization. E
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