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

J Mol Biol. 1999 Oct 8;292(5):1095-110. doi: 10.1006/jmbi.1999.3140.


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.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alanine / chemistry
  • Alanine / genetics*
  • Alanine / metabolism
  • Amino Acid Sequence
  • Amino Acid Substitution*
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Carrier Proteins / chemistry
  • DNA / genetics
  • DNA / metabolism*
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Dimerization
  • Hydrogen Bonding
  • Integration Host Factors
  • Models, Molecular
  • Molecular Sequence Data
  • Nuclear Magnetic Resonance, Biomolecular
  • PII Nitrogen Regulatory Proteins
  • Peptide Fragments / chemistry*
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Protein Structure, Secondary
  • Salmonella typhimurium / chemistry*
  • Solutions
  • Trans-Activators*
  • Transcription Factors*


  • Bacterial Proteins
  • Carrier Proteins
  • DNA-Binding Proteins
  • Integration Host Factors
  • PII Nitrogen Regulatory Proteins
  • Peptide Fragments
  • Solutions
  • Trans-Activators
  • Transcription Factors
  • DNA
  • Alanine