MgATP binding and hydrolysis determinants of NtrC, a bacterial enhancer-binding protein

When phosphorylated, the dimeric form of nitrogen regulatory protein C (Ntr C) of Salmonella typhimurium forms a larger oligomer(s) that can hydrolyze ATP and hence activate transcription by the sigma(54)-holoenzyme form of RN A polymerase. Studies of Mg-nucleoside triphosphate binding using a filter- binding assay indicated that phosphorylation is not required for nucleotide binding but probably controls nucleotide hydrolysis per se. Studies of bin ding by isothermal titration calorimetry indicated that the apparent K-d of unphosphorylated NtrC for MgATP gamma S is 100 mu M at 25 degrees C, and s tudies by filter binding indicated that the concentration of MgATP required for half-maximal binding is 130 mu M at 37 degrees C. Filter-binding studi es with mutant forms of NtrC defective in ATP hydrolysis implicated two reg ions of its central domain directly in nucleotide binding and three additio nal regions in hydrolysis. All five are highly conserved among activators o f sigma(54)-holoenzyme. Regions implicated in binding are the Walker A moti f and the region around residues G355 to R358, which may interact with the nucleotide base. Regions implicated in nucleotide hydrolysis are residues S 207 and E208, which have been proposed to lie in a region analogous to the switch I effector region of p21(ras) and other purine nucleotide-binding pr oteins; residue R294, which may be a catalytic residue; and residue D239, w hich is the conserved aspartate in the putative Walker B motif. D239 appear s to play a role in binding the divalent cation essential for nucleotide hy drolysis. Electron paramagnetic resonance analysis of Mn2+ binding indicate d that the central domain of NtrC does not bind divalent cation strongly in the absence of nucleotide.