Structural characterization of the N-terminal oligomerization domain of the bacterial chromatin-structuring protein, H-NS.

The H-NS protein plays a key role in condensing DNA and modulating gene expression in bacterial nucleoids. The mechanism by which this is achieved is dependent, at least in part, on the oligomerization of the protein. H-NS consists of two distinct domains; the N-terminal domain responsible for protein oligomerization, and the C-terminal DNA binding domain, which are separated by a flexible linker region. We present a multidimensional NMR study of the amino-terminal 64 residues of H-NS (denoted H-NS1-64) from Salmonella typhimurium, which constitute the oligomerization domain. This domain exists as a homotrimer, which is predicted to be self-associated through a coiled-coil configuration. NMR spectra show an equivalent magnetic environment for each monomer indicating that the polypeptide chains are arranged in parallel with complete 3-fold symmetry. Despite the limited resonance dispersion, an almost complete backbone assignment for 1H(N), 1H(alpha), 15N, 13CO and 13C(alpha) NMR resonances was obtained using a suite of triple resonance experiments applied to uniformly 15N-, 13C/15N- and 2H/13C/15N-labelled H-NS1-64 samples. The secondary structure of H-NS1-64 has been identified on the basis of the analysis of 1H(alpha), 13C(alpha), 13Cbeta and 13CO chemical shifts, NH/solvent exchange rates, intra-chain H(N)-H(N) and medium-range nuclear Overhauser enhancements (NOEs). Within the context of the homotrimer, each H-NS1-64 protomer consists of three alpha-helices spanning residues 2-8, 12-20 and 22-53, respectively. A topological model is presented for the symmetric H-NS1-64 trimer based upon the combined analysis of the helical elements and the pattern of backbone amide group 15N nuclear relaxation rates within the context of axially asymmetric diffusion tensor. In this model, the longest of the three helices (helix 3, residues 22-53) forms a coiled-coil interface with the other chains in the homotrimer. The two shorter N-terminal helices fold back onto the outer surface of the coiled-coil core and potentially act to stabilise this configuration.

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

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

[3]  C. Higgins,et al.  Oligomerization of the chromatin‐structuring protein H‐NS , 2000, Molecular microbiology.

[4]  A. Szabó,et al.  Model-free approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules. 1. Theory and range of validity , 1982 .

[5]  L. Pearl,et al.  Crystal structure and induction mechanism of AmiC–AmiR: a ligand‐regulated transcription antitermination complex , 1999, The EMBO journal.

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

[7]  L. Kay,et al.  Global folds of highly deuterated, methyl-protonated proteins by multidimensional NMR. , 1997, Biochemistry.

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

[9]  G. Siegal,et al.  Backbone dynamics of the C-terminal SH2 domain of the p85alpha subunit of phosphoinositide 3-kinase: effect of phosphotyrosine-peptide binding and characterization of slow conformational exchange processes. , 2000, Journal of molecular biology.

[10]  Ad Bax,et al.  Correlating Backbone Amide and Side-Chain Resonances in Larger Proteins By Multiple Relayed Triple Resonance NMR , 1992 .

[11]  D. S. Garrett,et al.  Defining long range order in NMR structure determination from the dependence of heteronuclear relaxation times on rotational diffusion anisotropy , 1997, Nature Structural Biology.

[12]  Annick Spassky,et al.  H1a, an E. coli DNA-binding protein which accumulates in stationary phase, strongly compacts DNA in vitro , 1984, Nucleic Acids Res..

[13]  D. States,et al.  A two-dimensional nuclear overhauser experiment with pure absorption phase in four quadrants☆ , 1982 .

[14]  A. Bax,et al.  Rotational diffusion anisotropy of human ubiquitin from 15N NMR relaxation , 1995 .

[15]  P. J. Kraulis,et al.  ANSIG: A program for the assignment of protein 1H 2D NMR spectra by interactive computer graphics , 1989, Journal of Magnetic Resonance (1969).

[16]  D. Ussery,et al.  The chromatin-associated protein H-NS. , 1994, Biochimie.

[17]  A. Gronenborn,et al.  Detection of nuclear Overhauser effects between degenerate amide proton resonances by heteronuclear three-dimensional NMR spectroscopy , 1990 .

[18]  H. Carr,et al.  The Principles of Nuclear Magnetism , 1961 .

[19]  A. Bax,et al.  Delineation of .alpha.-helical domains in deuteriated Staphylococcal nuclease by 2D NOE NMR spectroscopy , 1988 .

[20]  G. Bodenhausen,et al.  Natural abundance nitrogen-15 NMR by enhanced heteronuclear spectroscopy , 1980 .

[21]  L. Kay,et al.  Pulse sequences for removal of the effects of cross correlation between dipolar and chemical-shift anisotropy relaxation mechanisms on the measurement of heteronuclear T1 and T2 values in proteins , 1992 .

[22]  Weontae Lee,et al.  A Suite of Triple Resonance NMR Experiments for the Backbone Assignment of 15N, 13C, 2H Labeled Proteins with High Sensitivity , 1994 .

[23]  K. Drlica,et al.  Histonelike proteins of bacteria. , 1987, Microbiological reviews.

[24]  G. Lipari.  Model-free approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules , 1982 .

[25]  T. Mizuno,et al.  Solution structure of the DNA binding domain of a nucleoid‐associated protein, H‐NS, from Escherichia coli , 1995, FEBS letters.

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

[27]  L. Kay,et al.  Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy: application to staphylococcal nuclease. , 1989, Biochemistry.

[28]  C. Higgins,et al.  Protein H1: a role for chromatin structure in the regulation of bacterial gene expression and virulence? , 1990, Molecular microbiology.

[29]  T. Yamazaki,et al.  Identification of the DNA binding surface of H‐NS protein from Escherichia coli by heteronuclear NMR spectroscopy , 1999, FEBS letters.

[30]  A. Danchin,et al.  Role of Escherichia coli histone-like nucleoid-structuring protein in bacterial metabolism and stress response--identification of targets by two-dimensional electrophoresis. , 1997, European journal of biochemistry.

[31]  E. Zuiderweg,et al.  Use of13C-13C NOE for the assignment of NMR lines of larger labeled proteins at larger magnetic fields , 1996 .

[32]  K Wüthrich,et al.  A two-dimensional nuclear Overhauser enhancement (2D NOE) experiment for the elucidation of complete proton-proton cross-relaxation networks in biological macromolecules. , 1980, Biochemical and biophysical research communications.

[33]  A. J. Shaka,et al.  A Simple Windowless Mixing Sequence to Suppress Cross Relaxation in TOCSY Experiments , 1993 .

[34]  T. Mizuno,et al.  Clarification of the dimerization domain and its functional significance for the Escherichia coli nucleoid protein H-NS. , 1997, Journal of molecular biology.

[35]  G. Marius Clore,et al.  Determining the Magnitude of the Fully Asymmetric Diffusion Tensor from Heteronuclear Relaxation Data in the Absence of Structural Information , 1998 .

[36]  H. Buc,et al.  Probing the structure, function, and interactions of the Escherichia coli H-NS and StpA proteins by using dominant negative derivatives , 1996, Journal of bacteriology.

[37]  F. Richards,et al.  NMR sequential assignment of Escherichia coli thioredoxin utilizing random fractional deuteriation. , 1988, Biochemistry.

[38]  Paul A. Keifer,et al.  Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity , 1992 .

[39]  F. Dahlquist,et al.  Three-dimensional 13C-resolved proton NOE spectroscopy of uniformly 13C-labeled proteins for the NMR assignment and structure determination of larger molecules , 1990 .

[40]  A. Lupas.  Coiled coils: new structures and new functions. , 1996, Trends in biochemical sciences.

[41]  T. Mizuno,et al.  Systematic mutational analysis revealing the functional domain organization of Escherichia coli nucleoid protein H-NS. , 1996, Journal of molecular biology.

[42]  Richard R. Ernst,et al.  Coherence transfer by isotropic mixing: Application to proton correlation spectroscopy , 1983 .

[43]  Ad Bax,et al.  A powerful method of sequential proton resonance assignment in proteins using relayed 15N‐1H multiple quantum coherence spectroscopy , 1989, FEBS letters.

[44]  A. Danchin,et al.  Mutations in bglY, the structural gene for the DNA-binding protein H1, affect expression of several Escherichia coli genes. , 1990, Biochimie.

[45]  M. Wittekind,et al.  HNCACB, a High-Sensitivity 3D NMR Experiment to Correlate Amide-Proton and Nitrogen Resonances with the Alpha- and Beta-Carbon Resonances in Proteins , 1993 .

[46]  A. Bax,et al.  Protein backbone angle restraints from searching a database for chemical shift and sequence homology , 1999, Journal of biomolecular NMR.

[47]  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 .