Comparison of histone-like HU protein DNA-binding properties and HU/IHF protein sequence alignment.
ABSTRACT: The structure and function of bacterial nucleoid are controlled by histone-like proteins of HU/IHF family, omnipresent in bacteria and also founding archaea and some eukaryotes.HU protein binds dsDNA without sequence specificity and avidly binds DNA structures with propensity to be inclined such as forks, three/four-way junctions, nicks, overhangs and DNA bulges. Sequence comparison of thousands of known histone-like proteins from diverse bacteria phyla reveals relation between HU/IHF sequence, DNA-binding properties and other protein features.Performed alignment and clusterization of the protein sequences show that HU/IHF family proteins can be unambiguously divided into three groups, HU proteins, IHF_A and IHF_B proteins. HU proteins, IHF_A and IHF_B proteins are further partitioned into several clades for IHF and HU; such a subdivision is in good agreement with bacterial taxonomy. We also analyzed a hundred of 3D fold comparative models built for HU sequences from all revealed HU clades. It appears that HU fold remains similar in spite of the HU sequence variations. We studied DNA-binding properties of HU from N. gonorrhoeae, which sequence is similar to one of E.coli HU, and HU from M. gallisepticum and S. melliferum which sequences are distant from E.coli protein. We found that in respect to dsDNA binding, only S. melliferum HU essentially differs from E.coli HU. In respect to binding of distorted DNA structures, S. melliferum HU and E.coli HU have similar properties but essentially different from M. gallisepticum HU and N. gonorrhea HU. We found that in respect to dsDNA binding, only S. melliferum HU binds DNA in non-cooperative manner and both mycoplasma HU bend dsDNA stronger than E.coli and N. gonorrhoeae. In respect to binding to distorted DNA structures, each HU protein has its individual profile of affinities to various DNA-structures with the increased specificity to DNA junction.HU/IHF family proteins sequence alignment and classification are updated. Comparative modeling demonstrates that HU protein 3D folding's even more conservative than HU sequence. For the first time, DNA binding characteristics of HU from N. gonorrhoeae, M. gallisepticum and S. melliferum are studied. Here we provide detailed analysis of the similarity and variability of DNA-recognizing and bending of four HU proteins from closely and distantly related HU clades.
Project description:HU and IHF are members of a family of prokaryotic proteins that interact with the DNA minor groove in a sequence-specific (IHF) or non-specific (HU) manner to induce and/or stabilize DNA bending. HU plays architectural roles in replication initiation, transcription regulation and site-specific recombination, and is associated with bacterial nucleoids. Cocrystal structures of Anabaena HU bound to DNA (1P71, 1P78, 1P51) reveal that while underlying proline intercalation and asymmetric charge neutralization mechanisms of DNA bending are similar for IHF and HU, HU stabilizes different DNA bend angles ( approximately 105-140 degrees ). The two bend angles within a single HU complex are not coplanar, and the resulting dihedral angle is consistent with negative supercoiling. Comparison of HU-DNA and IHF-DNA structures suggests that sharper bending is correlated with longer DNA binding sites and smaller dihedral angles. An HU-induced bend may be better modeled as a hinge, not a rigid bend. The ability to induce or stabilize varying bend angles is consistent with HU's role as an architectural cofactor in many different systems that may require differing geometries.
Project description:IHF and HU are two heterodimeric nucleoid-associated proteins (NAP) that belong to the same protein family but interact differently with the DNA. IHF is a sequence-specific DNA-binding protein that bends the DNA by over 160°. HU is the most conserved NAP, which binds non-specifically to duplex DNA with a particular preference for targeting nicked and bent DNA. Despite their importance, the in vivo interactions of the two proteins to the DNA remain to be described at a high resolution and on a genome-wide scale. Further, the effects of these proteins on gene expression on a global scale remain contentious. Finally, the contrast between the functions of the homo- and heterodimeric forms of proteins deserves the attention of further study. Here we present a genome-scale study of HU- and IHF binding to the Escherichia coli K12 chromosome using ChIP-seq. We also perform microarray analysis of gene expression in single- and double-deletion mutants of each protein to identify their regulons. The sequence-specific binding profile of IHF encompasses ?30% of all operons, though the expression of <10% of these is affected by its deletion suggesting combinatorial control or a molecular backup. The binding profile for HU is reflective of relatively non-specific binding to the chromosome, however, with a preference for A/T-rich DNA. The HU regulon comprises highly conserved genes including those that are essential and possibly supercoiling sensitive. Finally, by performing ChIP-seq experiments, where possible, of each subunit of IHF and HU in the absence of the other subunit, we define genome-wide maps of DNA binding of the proteins in their hetero- and homodimeric forms.
Project description:HU and IHF are prokaryotic proteins that induce very large bends in DNA. They are present in high concentrations in the bacterial nucleoid and aid in chromosomal compaction. They also function as regulatory cofactors in many processes, such as site-specific recombination and the initiation of replication and transcription. HU and IHF have become paradigms for understanding DNA bending and indirect readout of sequence. While IHF shows significant sequence specificity, HU binds preferentially to certain damaged or distorted DNAs. However, none of the structurally diverse HU substrates previously studied in vitro is identical with the distorted substrates in the recently published Anabaena HU(AHU)-DNA cocrystal structures. Here, we report binding affinities for AHU and the DNA in the cocrystal structures. The binding free energies for formation of these AHU-DNA complexes range from approximately 10-14.5 kcal/mol, representing K(d) values in the nanomolar to low picomolar range, and a maximum stabilization of at least approximately 6.3 kcal/mol relative to complexes with undistorted, non-specific DNA. We investigated IHF binding and found that appropriate structural distortions can greatly enhance its affinity. On the basis of the coupling of structural and relevant binding data, we estimate the amount of conformational strain in an IHF-mediated DNA kink that is relieved by a nick (at least 0.76 kcal/mol) and pinpoint the location of the strain. We show that AHU has a sequence preference for an A+T-rich region in the center of its DNA-binding site, correlating with an unusually narrow minor groove. This is similar to sequence preferences shown by the eukaryotic nucleosome.
Project description:Here we report bisphenol derivatives of fluorene (BDFs) as a new type of chemical probes targeting a histone-like HU protein, a global regulator of bacterial nucleoids, via its dimerization interface perturbation. BDFs were identified by virtual screening and molecular docking that targeted the core of DNA-binding ?-saddle-like domain of the HU protein from Spiroplasma melliferum. However, NMR spectroscopy, complemented with molecular dynamics and site-directed mutagenesis, indicated that the actual site of the inhibitors' intervention consists of residues from the ?-helical domain of one monomer and the side portion of the DNA-binding domain of another monomer. BDFs inhibited DNA-binding properties of HU proteins from mycoplasmas S. melliferum, Mycoplasma gallicepticum and Escherichia coli with half-maximum inhibitory concentrations in the range between 5 and 10 µM. In addition, BDFs demonstrated antimicrobial activity against mycoplasma species, but not against E. coli, which is consistent with the compensatory role of other nucleoid-associated proteins in the higher bacteria. Further evaluation of antimicrobial effects of BDFs against various bacteria and viruses will reveal their pharmacological potential, and the allosteric inhibition mode reported here, which avoids direct competition for the binding site with DNA, should be considered in the development of small molecule inhibitors of nucleoid-associated proteins as well as other types of DNA-binding multimeric proteins.
Project description:IHF and HU are two heterodimeric nucleoid associated proteins (NAP) that belong to the same protein family but interact differently with the DNA. IHF is a sequence-specific DNA-binding protein that bends the DNA by over 160o. HU is the most conserved NAP which binds non-specifically to duplex DNA with a particular preference for targeting nicked and bent DNA. Despite their importance, the in-vivo interactions of the two proteins to the DNA remain to be described at a high resolution and on a genome-wide scale. Further, the effects of these proteins on gene expression on a global scale remain contentious. Finally, the contrast between the functions of the homo- and heterodimeric forms of these proteins deserves the attention of further study. Here we present a genome-scale study of HU- and IHF-binding to the E. coli K12 chromosome using ChIP-seq. We also perform microarray analysis of gene expression in single- and double-deletion mutants of each protein to identify their regulons. This data is for microarrays measuring gene expression changes in the various ihf mutants when compared to the wildtype.<br>
Project description:In prokaryotic cells, genomic DNA forms an aggregated structure with various nucleoid-associated proteins (NAPs). The functions of genomic DNA are cooperatively modulated by NAPs, of which HU is considered to be one of the most important. HU binds double-stranded DNA (dsDNA) and serves as a structural modulator in the genome architecture. It plays important roles in diverse DNA functions, including replication, segregation, transcription and repair. Interestingly, it has been reported that HU also binds single-stranded DNA (ssDNA) regardless of sequence. However, structural analysis of HU with ssDNA has been lacking, and the functional relevance of this binding remains elusive. In this study, we found that ssDNA induced a significant change in the secondary structure of Thermus thermophilus HU (TtHU), as observed by analysis of circular dichroism spectra. Notably, this change in secondary structure was sequence specific, because the complementary ssDNA or dsDNA did not induce the change. Structural analysis using nuclear magnetic resonance confirmed that TtHU and this ssDNA formed a unique structure, which was different from the previously reported structure of HU in complex with dsDNA. Our data suggest that TtHU undergoes a distinct structural change when it associates with ssDNA of a specific sequence and subsequently exerts a yet-to-be-defined function.
Project description:HU proteins belong to the nucleoid-associated proteins (NAPs) that are involved in vital processes such as DNA compaction and reparation, gene transcription etc. No data are available on the structures of HU proteins from mycoplasmas. To this end, the HU protein from the parasitic mycoplasma Spiroplasma melliferum KC3 was cloned, overexpressed in Escherichia coli and purified to homogeneity. Prismatic crystals of the protein were obtained by the vapour-diffusion technique at 4°C. The crystals diffracted to 1.36 Å resolution (the best resolution ever obtained for a HU protein). The diffraction data were indexed in space group C2 and the structure of the protein was solved by the molecular-replacement method with one monomer per asymmetric unit.
Project description:Legionella pneumophila is an intracellular parasite of protozoa that differentiates late in infection into metabolically dormant cysts that are highly infectious. Regulation of this process is poorly understood. Here we report that the small DNA binding regulatory proteins integration host factor (IHF) and HU are reciprocally expressed over the developmental cycle, with HU expressed during exponential phase and IHF expressed postexponentially. To assess the role of these regulatory proteins in development, chromosomal deletions were constructed. Single (ihfA or ihfB) and double deletion (Deltaihf) IHF mutants failed to grow in Acanthamoeba castellanii unless complemented in trans when expressed temporally from the ihfA promoter but not under P(tac) (isopropyl-beta-d-thiogalactopyranoside). In contrast, IHF mutants were infectious for HeLa cells, though electron microscopic examination revealed defects in late-stage cyst morphogenesis (thickened cell wall, intracytoplasmic membranes, and inclusions of poly-beta-hydroxybutyrate), and were depressed for the developmental marker MagA. Green fluorescent protein promoter fusion assays indicated that IHF and the stationary-phase sigma factor RpoS were required for full postexponential expression of magA. Finally, defects in cyst morphogenesis noted for Deltaihf mutants in HeLa cells correlated with a loss of both detergent resistance and hyperinfectivity compared with results for wild-type cysts. These studies establish IHF and HU as markers of developmental stages and show that IHF function is required for both differentiation and full virulence of L. pneumophila in natural amoebic hosts.
Project description:The heterodimeric integration host factor (IHF) is a site-specific DNA-binding and DNA-bending protein from Escherichia coli. It plays essential roles in a variety of DNA transactions including recombination, transcription and DNA replication. IHF's ability for concerted binding and bending of DNA is key to its biological function. Here we report the design, characterization and application of a single polypeptide chain IHF, termed scIHF2. In a novel approach for protein engineering, we inserted almost the entire alpha-subunit of IHF into the beta-subunit. DNA binding and DNA bending assays revealed that purified wild-type IHF and scIHF2 behave very similarly. Further, scIHF2 is required for site-specific integrative recombination by phage lambda integrase and for pSC101 replication in a DeltaIHF E.coli host. It also triggers site-specific integrative and excisive recombination in vitro to the same extent as the wild-type protein. We also demonstrate that scIHF2 is stably expressed in HeLa cells, that it is localized primarily in the cell nucleus and that it triggers integrative recombination in mammalian cells by wild-type integrase. Hence, scIHF2 may be used as a novel regulatory cofactor for recombination or other DNA transactions in mammalian cells that require or benefit from sequence-specific high precision DNA bending.
Project description:The Escherichia coli chromosome is condensed into an ill-defined structure known as the nucleoid. Nucleoid-associated DNA-binding proteins are involved in maintaining this structure and in mediating chromosome compaction. We have exploited chromatin immunoprecipitation and high-density microarrays to study the binding of three such proteins, FIS, H-NS and IHF, across the E.coli genome in vivo. Our results show that the distribution of these proteins is biased to intergenic parts of the genome, and that the binding profiles overlap. Hence some targets are associated with combinations of bound FIS, H-NS and IHF. In addition, many regions associated with FIS and H-NS are also associated with RNA polymerase.