Comparison of histone-like HU protein DNA-binding properties and HU/IHF protein sequence alignment

<div><p>Background</p><p>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.</p><p>Methodology and principal findings</p><p>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 <i>N</i>. <i>gonorrhoeae</i>, which sequence is similar to one of <i>E</i>.<i>coli</i> HU, and HU from <i>M</i>. <i>gallisepticum</i> and <i>S</i>. <i>melliferum</i> which sequences are distant from <i>E</i>.<i>coli</i> protein. We found that in respect to dsDNA binding, only <i>S</i>. <i>melliferum</i> HU essentially differs from <i>E</i>.<i>coli</i> HU. In respect to binding of distorted DNA structures, <i>S</i>. <i>melliferum</i> HU and <i>E</i>.<i>coli</i> HU have similar properties but essentially different from <i>M</i>. <i>gallisepticum</i> HU and <i>N</i>. <i>gonorrhea</i> HU. We found that in respect to dsDNA binding, only <i>S</i>. <i>melliferum</i> HU binds DNA in non-cooperative manner and both mycoplasma HU bend dsDNA stronger than <i>E</i>.<i>coli</i> and <i>N</i>. <i>gonorrhoeae</i>. 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.</p><p>Conclusions and significance</p><p>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 <i>N</i>. <i>gonorrhoeae</i>, <i>M</i>. <i>gallisepticum</i> and <i>S</i>. <i>melliferum</i> 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.</p></div

Model of <i>Pseudomonas syringae</i> HU (magenta) superimposed with <i>E</i>. <i>coli</i> HUα model (cyan).

<p>Angle between alpha helix 1 and alpha helix 2 is shown in green.</p

HU binding to dsDNA of various lengths.

<p>Binding of labeled DNA to HU proteins was analyzed by polyacrylamide gel electrophoresis. The gel was buffered with 50 mM Tris–borate; binding mixture contains 40 mM NaCl. DNA samples were: dsDNA of sequence ‘D’ with the length varying from 21 to 48 bp (indicated at the bottom). HU origin and concentration is indicated at the top (“-“, no HU was added). Bands corresponding to HU-DNA complexes are marked with arrows, the number of HU dimers in each complex is indicated on the left of the arrow. Panels correspond to HU proteins of various bacteria, protein concentrations are indicated.</p

Model of HU <i>Bifidobacterium longum</i> from Actinobacteria (clade HU_acti_0), superimposed with <i>E</i>. <i>coli</i> HUα model.

<p><i>B</i>. <i>longum</i> HU is shown in yellow, <i>E</i>. <i>coli</i> HUα—in cyan. Angle between alpha helix 2 and alpha helix 3 is shown in magenta.</p

PCA plot of the location of aligned sequences of HU/IHF proteins on three axes.

<p>Three main groups of proteins, HU, IHF_A and IHF_B, are indicated as well as results of further subdivision of the protein sequences (HU and IHF clades).<b>A.</b> Most populated HU clades: clade HU_Firmicutes (mainly originated from HU of Firmicutes species) and clades HU_ecoA and HU_ecoB (mainly from proteobacteria) are shown in magenta, bleu and cyan, respectively. Other apparent HU/IHF clades are indicated. Position HUs and IHFs proteins of <i>E</i>. <i>coli</i> are shown in red, as well as positions of HU of <i>N</i>. <i>gonorrhoeae</i> (NG), <i>M</i>. <i>gallisepticum</i> (MG), and <i>S</i>. <i>melliferum</i> (SM). <i>E</i>. <i>coli</i> HUα and Huβ are very close to each other (62 identities in 90 amino acid core sequence), and IHFα and IHFβ are far from each other (24 identities of 90). We believe that it is the reason why HU separation onto two groups, one close to HUα, and another close to HUβ, is ambiguous. <b>B.</b> IHF clades. Result of two major IHF group subdivisions shown in color: IHF_A magenta, cyan and green, IHF_B red, yellow, and blue. HU sequences are shown in grey.</p

HU binding to “distorted” DNA structures checked by polyacrylamide gel mobility assay.

<p>HU protein at concentrations indicated above the gel image (“-“, no HU was added) was mixed with 5’-labelled DNA in a buffer containing 150 mM NaCl; the bound and free DNA were gel-separated. DNA structures indicated at the bottom of the gel images: n, nicked DNA; ds, dsDNA; A1, A3 and A7, DNA bulges, containing one, three or seven non-paired adenines in one of DNA strands; J–four-way junction; fork, ssDNA fork; ov, DNA overhang; iJ, incomplete junction lacking one DNA strand; inv, DNA invasion. Panels correspond to HU proteins of various bacteria.</p

Model of <i>E</i>.<i>coli</i> HUα dimer with hotspots for amino acid insertions and deletions.

<p>Each HU monomer contains three alpha helixes and five beta strands. HU body (helixes 1 and 2) is responsible for dimer stabilization. HU arms are responsible for DNA binding. Hotspots for amino acid insertions and deletions in HU are shown.</p

Consensus sequences of three IHF_A, three IHF_B, and several HU clades revealed in this study.

<p>Conservative residues are highlighted. Alpha helixes and beta sheets are indicated with yellow and blue blocks, respectively.</p

Model of <i>E</i>.<i>coli</i> HUα dimer with distances between C-alpha atoms that were measured.

<p>Positions of amino acid residues corresponding to the C-termini of alpha helixes 2 are indicated and distance between corresponding C-alpha atoms of both monomers is shown with khaki dotted line. Positions of the amino acid residues at the C-termini of beta strands 2 and N-termini of beta strands 5 as well as their neighboring residues are indicated and the corresponding distances between C-alpha atoms of both monomers are shown with rose and blue dotted line, respectively.</p

The profiles of HU affinities to various DNA-structures.

<p>Each column represents the association constant of HU complex with one DNA structure (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188037#pone.0188037.g010" target="_blank">Fig 10</a> legend for structures description) normalized on the association constants of HU-dsDNA complex of the same protein. Data from at least three independent experiments were combined.</p