Solution structure of the DNA-binding domain of NtrC with three alanine substitutions

The structure of the 20 kDa C-terminal DNA-binding domain of NtrC from Salm onella typhimurium (residues Asp380-Glu469) with alanine replacing Arg456,A sn457, and Arg461, was determined by NMR spectroscopy. NtrC is a homodimeri c enhancer-binding protein that activates the transcription of genes whose products are required for nitrogen metabolism. The 91-residue C-terminal do main contains the determinants necessary for dimerization and DNA-binding o f the full length protein. The mutant protein does not bind to DNA but reta ins many characteristics of the wild-type protein, and the mutant domain ex presses at high yield (20 mg/l) in minimal medium. Three-dimensional H-1/C-13/N-15 triple-resonance, H-1-C-13-C-13-H-1 correla tion and N-15-separated nuclear Overhauser effect (NOE) spectroscopy experi ments were used to make backbone and side-chain H-1, N-15, and C-13 assignm ents. The structures were calculated using a total of 1580 intra and inter- monomer distance and hydrogen bond restraints (88 hydrogen bonds; 44 hydrog en bond restraints), and 88 phi dihedral restraints for residues Asp400 thr ough Glu469 in both monomers. A total of 54 ambiguous restraints (intra or inter-monomer) involving residues close to the 2-fold symmetry axis were al so included. Each monomer consists of four helical segments. Helices A (Trp402-Leu414) a nd B (Leu421-His440) join with those of another monomer to form an antipara llel four-helix bundle. Helices C (Gln446-Leu451) and D (Ala456-Met468) of each monomer adopt a classic helix-turn-helix DNA-binding fold at either en d of the protein. The backbone rms deviation for the 28 best of 40 starting structures is 0.6(+/- 0.2) Angstrom. Structural differences between the C- terminal domain of NtrC and the homologous Factor for Inversion Stimulation are discussed. (C) 1999 Academic Press.