Oligomerization of the chromatin-structuring protein H-NS

H-NS is a major component of the bacterial nucleoid, involved in condensing and packaging DNA and modulating gene expression. The mechanism by which this is achieved remains unclear. Genetic data show that the biological properties of H-NS are influenced by its oligomerization properties. We have applied a variety of biophysical techniques to study the structural basis of oligomerization of the H-NS protein from Salmonella typhimurium. The N-terminal 89 amino acids are responsible for oligomerization. The first 64 residues form a trimer dominated by an alpha-helix, likely to be in coiled-coil conformation. Extending this polypeptide to 89 amino acids generated higher order, heterodisperse oligomers. Similarly, in the full-length protein no single, defined oligomeric state is adopted. The C-terminal 48 residues do not participate in oligomerization and form a monomeric, DNA-binding domain. These N- and C-terminal domains are joined via a flexible linker which enables them to function independently within the context of the full-length protein. This novel mode of oligomerization may account for the unusual binding properties of H-NS.     

The H-NS dimerization domain defines a new fold contributing to DNA recognition

H-NS, a protein found in Gram-negative bacteria, is involved in structuring the bacterial chromosome and acts as a global regulator for the expression of a wide variety of genes. These functions are correlated with both its DNA-binding and oligomerization properties. We have identified the minimal dimerization domain of H-NS, a 46 amino acid-long N-terminal fragment, and determined its structure using heteronuclear NMR spectroscopy. The highly intertwined structure of the dimer, reminiscent of a handshake, defines a new structural fold, which may offer a possibility for discriminating prokaryotic from eukaryotic proteins in drug design. Using mutational analysis, we also show that this N-terminal domain actively contributes to DNA binding, conversely to the current paradigm. Together, our data allows us to propose a model for the action of full length H-NS.     

Identification of a regulatory protein required for pressure-responsive gene expression in the deep-sea bacterium Photobacterium species strain SS9

Here, we report the characterization of a gene necessary for hydrostatic pressure regulation of gene expression in the deep-sea bacterium Photobacterium species strain SS9. The deduced amino acid sequence of the gene product shares extensive similarity to ToxR, a transmembrane DNA-binding protein first discovered as a virulence determinant in the pathogenic bacterium Vibrio cholerae . Changes in hydrostatic pressure induce changes in both the abundance and the activity of the SS9 ToxR protein (or the activity of a ToxR-regulated protein). As with other high-pressure-inducible phenomena observed in higher organisms, anaesthetics antagonize high-pressure signalling mediated by ToxR. It is suggested that SS9 ToxR has evolved the ability to respond to pressure-mediated alterations in membrane structure. V. cholerae and SS9 also share similarity in a ToxR-regulated protein, indicating that part of the ToxR regulon is conserved in diverse members of the family Vibrionaceae. The SS9 ToxR system represents a useful model for studies of signal transduction and environmental adaptation in the largest portion of the biosphere, the deep sea.     

Crystal structure of the N-terminal dimerisation domain of VicH, the H-NS-like protein of Vibrio cholerae

The histone-like nucleoid structuring (H-NS) protein is a global modulator of gene expression in Gram-negative bacteria. VicH, the H-NS protein of Vibrio cholerae, regulates the expression of certain major virulence determinants implicated in the pathogenesis of cholera. We present here the 2.5A crystal structure of the N-terminal oligomerisation domain of VicH (VicH_Nt). VicH_Nt adopts the same fold and dimeric assembly as the NMR structure of Escherichia coli H-NS_Nt, thus validating this fold against conflicting data. The structural similarity of V.cholerae VicH_Nt and E.coli H-NS_Nt, despite differences in origin, system of expression, experimental conditions and techniques used, indicates that the fold determined in our studies is robust to experimental conditions. Structural analysis and homology modelling were carried out to further elucidate the molecular basis of the functional polyvalence of the N-terminal domain. Our analysis of members of the H-NS superfamily supports the suggestion that the oligomerisation function of H-NS_Nt is conserved even in more distantly related proteins.     

New roles for key residues in helices H1 and H2 of the Escherichia coli H-NS N-terminal domain: H-NS dimer stabilization and Hha binding

Bacterial nucleoid-associated proteins H-NS and Hha modulate gene expression in response to environmental factors. The N-terminal domain of H-NS is involved in homomeric and heteromeric protein-protein interactions. Homomeric interaction leads to the formation of dimers and higher oligomers. Heteromeric interactions with Hha-like proteins modify the modulatory properties of H-NS. In this study, we have used NMR and mutagenesis of the N-terminal domain of H-NS to identify the Hha-binding region around helices H1 and H2 of H-NS. Two conserved arginine residues, R12 and R15, located in the same side and in adjacent turns of helix H2 are shown to be involved in two different protein-protein interactions: R12 is essential for Hha binding and does not affect H-NS dimer formation, and R15 does not affect Hha binding but is essential for the proper folding of H-NS dimers. Our results demonstrate a close structural connection between Hha-H-NS interactions and H-NS dimerization that may be involved in a possible mechanism for the modulation of the H-NS regulatory activity by Hha.     

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.     

Functional replacement of the oligomerization domain of H-NS by the Hha protein of Escherichia coli

Members of the H-NS family of proteins play a relevant role as modulators of gene expression in gram-negative bacteria. Interaction of these proteins with members of the Hha/YmoA family of proteins has been previously reported. It has been hypothesized that the latter proteins are functionally equivalent to the N-terminal domain of H-NS-like proteins. In this report we test this assumption by replacing the N-terminal domain of Escherichia coli H-NS by Hha. It has been possible to obtain a functional protein that can compensate for some of the hns-induced phenotypes. These results highlight the relevance of H-NS-Hha interactions to generate heterooligomeric complexes that modulate gene expression in gram-negative bacteria.     

A truncated H-NS-like protein from enteropathogenic Escherichia coli acts as an H-NS antagonist

The H-NS nucleoid-associated protein of Escherichia coli is the founder member of a widespread family of gene regulatory proteins which have a bipartite structure, consisting of an N-terminal coiled-coil oligomerization domain and a C-terminal DNA-binding domain. Here we characterize a family of naturally occurring truncated H-NS derivatives lacking the DNA-binding domain, which we term the H-NST family. H-NST proteins are found in large genomic islands in pathogenic E. coli strains, which are absent from the corresponding positions in the E. coli K-12 genome. Detailed analysis of the H-NST proteins from enteropathogenic E. coli (EPEC) and uropathogenic E. coli (UPEC) shows that the EPEC protein (H-NST(EPEC)) has a potent anti-H-NS function at the classical H-NS-repressed operon proU. This correlates with the ability of H-NST(EPEC) to co-purify with H-NS in vitro, and can be abolished by a mutation of leucine 30 to proline which is predicted to prevent the N-terminal region from forming a coiled-coil structure. In contrast, despite being 90% identical to H-NST(EPEC) at the protein level, the UPEC homologue (H-NST(UPEC)) has only a weak anti-H-NS activity, correlating with a much-reduced ability to interact with H-NS during column chromatography. A single amino acid difference at residue 16 appears to account for these different properties. The hnsT(EPEC) gene is transcribed monocistronically and expressed throughout the exponential growth phase in DMEM medium. Our data suggest that a truncated derivative of H-NS encoded by an ancestral mobile DNA element can interact with the endogenous H-NS regulatory network of a bacterial pathogen.