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 Salmonella typhimurium (residues Asp380-Glu469) with alanine replacing Arg456, Asn457, and Arg461, was determined by NMR spectroscopy. NtrC is a homodimeric enhancer-binding protein that activates the transcription of genes whose products are required for nitrogen metabolism. The 91-residue C-terminal domain contains the determinants necessary for dimerization and DNA-binding of the full length protein. The mutant protein does not bind to DNA but retains many characteristics of the wild-type protein, and the mutant domain expresses at high yield (20 mg/l) in minimal medium. Three-dimensional (1)H/(13)C/(15)N triple-resonance, (1)H-(13)C-(13)C-(1)H correlation and (15)N-separated nuclear Overhauser effect (NOE) spectroscopy experiments were used to make backbone and side-chain (1)H,(15)N, and (13)C assignments. The structures were calculated using a total of 1580 intra and inter-monomer distance and hydrogen bond restraints (88 hydrogen bonds; 44 hydrogen bond restraints), and 88 phi dihedral restraints for residues Asp400 through Glu469 in both monomers. A total of 54 ambiguous restraints (intra or inter-monomer) involving residues close to the 2-fold symmetry axis were also included. Each monomer consists of four helical segments. Helices A (Trp402-Leu414) and B (Leu421-His440) join with those of another monomer to form an antiparallel 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 end of the protein. The backbone rms deviation for the 28 best of 40 starting structures is 0.6 (+/-0.2) A. Structural differences between the C-terminal domain of NtrC and the homologous Factor for Inversion Stimulation are discussed.
Pelton
J G
JG
Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94710, USA.
Kustu
S
S
Wemmer
D E
DE
D000409:Alanine / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D006860:Hydrogen Bonding; D034982:Integration Host Factors; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D051922:PII Nitrogen Regulatory Proteins; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D012486:Salmonella typhimurium / Q000737:chemistry*; D012996:Solutions; D015534:Trans-Activators; D014157:Transcription Factors
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D034982:Integration Host Factors; D051922:PII Nitrogen Regulatory Proteins; D010446:Peptide Fragments; D012996:Solutions; D015534:Trans-Activators; D014157:Transcription Factors; D004247:DNA; D000409:Alanine