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.

[1]  M. Sporn,et al.  Transforming growth factor beta 1: three-dimensional structure in solution and comparison with the X-ray structure of transforming growth factor beta 2. , 1996, Biochemistry.

[2]  R. Dimitrov,et al.  The NMR solution structure and characterization of pH dependent chemical shifts of the β-elicitin, cryptogein , 1998 .

[3]  W. Gmeiner,et al.  Improved 3D gd-HCACO and gd-(H)CACO-TOCSY experiments for isotopically enriched proteins dissolved in H2O , 1996, Journal of biomolecular NMR.

[4]  W. Fairbrother,et al.  The solution structure of melanoma growth stimulating activity. , 1994, Journal of molecular biology.

[5]  F. Neidhardt,et al.  Escherichia Coli and Salmonella: Typhimurium Cellular and Molecular Biology , 1987 .

[6]  P. Matsumura,et al.  Restoration of flagellar clockwise rotation in bacterial envelopes by insertion of the chemotaxis protein CheY. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[7]  S W Englander,et al.  Isotope effects in peptide group hydrogen exchange , 1993, Proteins.

[8]  F. Ruscetti,et al.  28. – Transforming Growth Factor β1 , 1998 .

[9]  K. Wüthrich,et al.  Support of1H NMR assignments in proteins by biosynthetically directed fractional13C-labeling , 1992 .

[10]  S. Kustu,et al.  The bacterial enhancer-binding protein NtrC as a molecular machine. , 1998, Cold Spring Harbor symposia on quantitative biology.

[11]  T. Steitz,et al.  Crystal structure of a CAP-DNA complex: the DNA is bent by 90 degrees , 1991, Science.

[12]  M Ikura,et al.  Improved three-dimensional1H−13C−1H correlation spectroscopy of a13C-labeled protein using constant-time evolution , 1991, Journal of biomolecular NMR.

[13]  Ad Bax,et al.  Rapid recording of 2D NMR spectra without phase cycling. Application to the study of hydrogen exchange in proteins , 1989 .

[14]  A. Contreras,et al.  The effect on the function of the transcriptional activator NtrC from Klebsiella pneumoniae of mutations in the DNA-recognition helix. , 1988, Nucleic acids research.

[15]  M Nilges,et al.  A calculation strategy for the structure determination of symmetric demers by 1H NMR , 1993, Proteins.

[16]  A. J. Shaka,et al.  Computer-optimized decoupling scheme for wideband applications and low-level operation , 1985 .

[17]  R. C. Johnson,et al.  Identification of two functional regions in Fis: the N‐terminus is required to promote Hin‐mediated DNA inversion but not lambda excision. , 1991, The EMBO journal.

[18]  A. Petros,et al.  Solution structure of the DNA-binding domain of a human papillomavirus E2 protein: evidence for flexible DNA-binding regions. , 1996, Biochemistry.

[19]  Jack Greenblatt,et al.  Methods for Measurement of Intermolecular NOEs by Multinuclear NMR Spectroscopy: Application to a Bacteriophage λ N-Peptide/boxB RNA Complex , 1997 .

[20]  Ad Bax,et al.  An efficient experiment for sequential backbone assignment of medium-sized isotopically enriched proteins , 1992 .

[21]  Ad Bax,et al.  MLEV-17-based two-dimensional homonuclear magnetization transfer spectroscopy , 1985 .

[22]  A. Gronenborn,et al.  Complete resonance assignment for the polypeptide backbone of interleukin 1 beta using three-dimensional heteronuclear NMR spectroscopy. , 1990, Biochemistry.

[23]  Reid C. Johnson,et al.  The transactivation region of the Fis protein that controls site‐specific DNA inversion contains extended mobile β‐hairpin arms , 1997, The EMBO journal.

[24]  Yawen Bai,et al.  Primary structure effects on peptide group hydrogen exchange , 1993, Biochemistry.

[25]  A. J. Shaka,et al.  Iterative schemes for bilinear operators; application to spin decoupling , 1988 .

[26]  S. Grzesiek,et al.  Improved 3D triple-resonance NMR techniques applied to a 31 kDa protein , 1992 .

[27]  Dieter Suter,et al.  Two-dimensional chemical exchange and cross-relaxation spectroscopy of coupled nuclear spins , 1981 .

[28]  M. Karplus,et al.  CHARMM: A program for macromolecular energy, minimization, and dynamics calculations , 1983 .

[29]  S. Kustu,et al.  Prokaryotic enhancer-binding proteins reflect eukaryote-like modularity: the puzzle of nitrogen regulatory protein C , 1993, Journal of bacteriology.

[30]  A. Bax,et al.  Measurement of long-range 13C-13C J couplings in a 20-kDa protein-peptide complex , 1992 .

[31]  P. Domaille,et al.  Heteronuclear (1H, 13C, 15N) NMR assignments and solution structure of the monocyte chemoattractant protein-1 (MCP-1) dimer. , 1996, Biochemistry.

[32]  S. Kustu,et al.  Mutant forms of the enhancer-binding protein NtrC can activate transcription from solution. , 1997, Journal of molecular biology.

[33]  P. V. van Zijl,et al.  Improved sensitivity of HSQC spectra of exchanging protons at short interscan delays using a new fast HSQC (FHSQC) detection scheme that avoids water saturation. , 1995, Journal of magnetic resonance. Series B.

[34]  A. Bax,et al.  Measurement of Two- and Three-Bond Proton to Methyl-Carbon J Couplings in Proteins Uniformly Enriched with 13C , 1993 .

[35]  M Karplus,et al.  The three‐dimensional structure of α1‐purothionin in solution: combined use of nuclear magnetic resonance, distance geometry and restrained molecular dynamics , 1986, The EMBO journal.

[36]  W. Saenger,et al.  Crystal structure of the factor for inversion stimulation FIS at 2.0 A resolution. , 1992, Journal of molecular biology.

[37]  S. Grzesiek,et al.  NMRPipe: A multidimensional spectral processing system based on UNIX pipes , 1995, Journal of biomolecular NMR.

[38]  S. Kustu,et al.  Prokaryotic transcriptional enhancers and enhancer-binding proteins. , 1991, Trends in biochemical sciences.

[39]  P. Bork,et al.  Evolution of new protein function: recombinational enhancer Fis originated by horizontal gene transfer from the transcriptional regulator NtrC , 1998, FEBS letters.

[40]  The molecular structure of wild-type and a mutant Fis protein: relationship between mutational changes and recombinational enhancer function or DNA binding. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[41]  Vladimir Sklenar,et al.  Gradient-Tailored Water Suppression for 1H-15N HSQC Experiments Optimized to Retain Full Sensitivity , 1993 .

[42]  A. J. Shaka,et al.  Evaluation of a new broadband decoupling sequence: WALTZ-16 , 1983 .

[43]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[44]  D. Torchia,et al.  1H, 15N, and 13C NMR signal assignments of IIIGlc, a signal-transducing protein of Escherichia coli, using three-dimensional triple-resonance techniques. , 1991, Biochemistry.

[45]  R. C. Johnson,et al.  Identification of new Fis binding sites by DNA scission with Fis-1,10-phenanthroline-copper(I) chimeras. , 1996, Biochemistry.

[46]  L. Kay,et al.  New methods for the measurement of NHCαH coupling constants in 15N-labeled proteins , 1990 .

[47]  A. Wedel,et al.  Oligomerization of NTRC at the glnA enhancer is required for transcriptional activation. , 1993, Genes & development.

[48]  A. Ninfa,et al.  Initiation of transcription at the bacterial glnAp2 promoter by purified E. coli components is facilitated by enhancers , 1987, Cell.

[49]  G. Marius Clore,et al.  1H1H correlation via isotropic mixing of 13C magnetization, a new three-dimensional approach for assigning 1H and 13C spectra of 13C-enriched proteins , 1990 .

[50]  D. S. Garrett,et al.  Increased Resolution and Improved Spectral Quality in Four-Dimensional 13C/13C-Separated HMQC-NOESY-HMQC Spectra Using Pulsed Field Gradients , 1993 .

[51]  K. Wüthrich,et al.  Stereospecific nuclear magnetic resonance assignments of the methyl groups of valine and leucine in the DNA-binding domain of the 434 repressor by biosynthetically directed fractional 13C labeling. , 1989, Biochemistry.

[52]  A. Gronenborn,et al.  Analysis of the backbone dynamics of interleukin-8 by 15N relaxation measurements. , 1993, Journal of molecular biology.

[53]  T. Pawson,et al.  Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation. , 1994, Biochemistry.

[54]  A. Gronenborn,et al.  Solution structure of the N-terminal zinc binding domain of HIV-1 integrase , 1997, Nature Structural Biology.

[55]  Robert Powers,et al.  A common sense approach to peak picking in two-, three-, and four-dimensional spectra using automatic computer analysis of contour diagrams , 1991 .

[56]  C. Chothia.  Structural invariants in protein folding , 1975, Nature.

[57]  K. Wüthrich.  NMR of proteins and nucleic acids , 1988 .

[58]  A M Gronenborn,et al.  Four-dimensional heteronuclear triple-resonance NMR spectroscopy of interleukin-1 beta in solution. , 1990, Science.

[59]  D. Kern,et al.  Structural and functional analyses of activating amino acid substitutions in the receiver domain of NtrC: evidence for an activating surface. , 1997, Journal of molecular biology.

[60]  S. Kustu,et al.  Expression of sigma 54 (ntrA)-dependent genes is probably united by a common mechanism. , 1989, Microbiological reviews.

[61]  M. Billeter,et al.  Calibration of the angular dependence of the amide proton-Cα proton coupling constants, 3JHNα, in a globular protein: Use of 3JHNα for identification of helical secondary structure , 1984 .

[62]  M. Nilges,et al.  Calculation of symmetric multimer structures from NMR data using a priori knowledge of the monomer structure, co-monomer restraints, and interface mapping: The case of leucine zippers , 1996, Journal of biomolecular NMR.

[63]  L. Kay,et al.  Comparison of different modes of two-dimensional reverse-correlation NMR for the study of proteins , 1990 .

[64]  M. Billeter,et al.  MOLMOL: a program for display and analysis of macromolecular structures. , 1996, Journal of molecular graphics.

[65]  R. C. Johnson,et al.  The Fis protein: it's not just for DNA inversion anymore , 1992, Molecular microbiology.

[66]  D E Wemmer,et al.  Three-dimensional solution structure of the N-terminal receiver domain of NTRC. , 1995, Biochemistry.

[67]  J. Thornton,et al.  Helix geometry in proteins. , 1988, Journal of molecular biology.

[68]  J. Thornton,et al.  Stereochemical quality of protein structure coordinates , 1992, Proteins.

[69]  C. Pace,et al.  How to measure and predict the molar absorption coefficient of a protein , 1995, Protein science : a publication of the Protein Society.

[70]  G. Wagner,et al.  Structure and mobility of the PUT3 dimer , 1997, Nature Structural Biology.

[71]  R. C. Johnson,et al.  Variable structures of Fis-DNA complexes determined by flanking DNA-protein contacts. , 1996, Journal of molecular biology.

[72]  K Wüthrich,et al.  Pseudo-structures for the 20 common amino acids for use in studies of protein conformations by measurements of intramolecular proton-proton distance constraints with nuclear magnetic resonance. , 1983, Journal of molecular biology.

[73]  Eric Oldfield,et al.  1H, 13C and 15N chemical shift referencing in biomolecular NMR , 1995, Journal of biomolecular NMR.

[74]  G. Wagner,et al.  An optimized 3D NOESY-HSQC. , 1996, Journal of magnetic resonance. Series B.

[75]  J. F. Thompson,et al.  Empirical estimation of protein-induced DNA bending angles: applications to lambda site-specific recombination complexes. , 1988, Nucleic acids research.

[76]  J. Wootton,et al.  Sequence and domain relationships of ntrC and nifA from Klebsiella pneumoniae: homologies to other regulatory proteins. , 1986, The EMBO journal.

[77]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[78]  A. Bax,et al.  Resolution enhancement and spectral editing of uniformly 13C-enriched proteins by homonuclear broadband 13C decoupling , 1992 .

[79]  R. C. Johnson,et al.  The structure of Fis mutant Pro61Ala illustrates that the kink within the long alpha-helix is not due to the presence of the proline residue. , 1995, The Journal of biological chemistry.

[80]  B. Matthews,et al.  The helix-turn-helix DNA binding motif. , 1989, The Journal of biological chemistry.

[81]  A. Bax,et al.  Optimized recording of heteronuclear multidimensional NMR spectra using pulsed field gradients , 1992 .

[82]  C. Ball,et al.  Isolation of the gene encoding the Hin recombinational enhancer binding protein. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[83]  F. Ausubel,et al.  Two-component regulatory systems responsive to environmental stimuli share strongly conserved domains with the nitrogen assimilation regulatory genes ntrB and ntrC. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[84]  T F Havel,et al.  The solution structure of eglin c based on measurements of many NOEs and coupling constants and its comparison with X‐ray structures , 1992, Protein science : a publication of the Protein Society.

[85]  K. Wüthrich,et al.  Improved spectral resolution in cosy 1H NMR spectra of proteins via double quantum filtering. , 1983, Biochemical and biophysical research communications.

[86]  M. Rance,et al.  Suppression of cross-relaxation effects in TOCSY spectra via a modified DIPSI-2 mixing sequence , 1992 .

[87]  M Kokkinidis,et al.  Structure of the ColE1 rop protein at 1.7 A resolution. , 1987, Journal of molecular biology.

[88]  S. Kustu,et al.  Mechanism of Transcriptional Activation by NtrC , 1995 .

[89]  E. Morett,et al.  A proposed architecture for the central domain of the bacterial enhancer‐binding proteins based on secondary structure prediction and fold recognition , 1997, Protein science : a publication of the Protein Society.

[90]  S. Kustu,et al.  The major dimerization determinants of the nitrogen regulatory protein NTRC from enteric bacteria lie in its carboxy-terminal domain. , 1994, Journal of molecular biology.

[91]  M. Rooman,et al.  Structural classification of HTH DNA-binding domains and protein-DNA interaction modes. , 1996, Journal of molecular biology.

[92]  Ann M Stock,et al.  Two-component signal transduction. , 2000, Annual review of biochemistry.

[93]  C. Sander,et al.  The structure of ColE1 rop in solution , 1991, Journal of biomolecular NMR.