Structural insights into thrombolytic activity of destabilase from medicinal leech

Destabilase from the medical leech Hirudo medicinalis belongs to the family of i-type lysozymes. It has two different enzymatic activities: microbial cell walls destruction (muramidase activity), and dissolution of the stabilized fibrin (isopeptidase activity). Both activities are known to be inhibited by sodium chloride at near physiological concentrations, but the structural basis remains unknown. Here we present two crystal structures of destabilase, including a 1.1 Å-resolution structure in complex with sodium ion. Our structures reveal the location of sodium ion between Glu34/Asp46 residues, which were previously recognized as a glycosidase active site. While sodium coordination with these amino acids may explain inhibition of the muramidase activity, its influence on previously suggested Ser49/Lys58 isopeptidase activity dyad is unclear. We revise the Ser49/Lys58 hypothesis and compare sequences of i-type lysozymes with confirmed destabilase activity. We suggest that the general base for the isopeptidase activity is His112 rather than Lys58. pKa calculations of these amino acids, assessed through the 1 μs molecular dynamics simulation, confirm the hypothesis. Our findings highlight the ambiguity of destabilase catalytic residues identification and build foundations for further research of structure–activity relationship of isopeptidase activity as well as structure-based protein design for potential anticoagulant drug development.</p

Productivity of breeding pigs during marl feeding in areas with high density of soil pollution with radiocesium

The paper considers the experimentally confirmed data on marl efficiency in the zones with increased level of radionuclides in the soil (5–10 and 15–40 Ki/km2) in the last third of female pig pregnancy in the amount of 2.0 % of the diet dry matter. Feeding with local mineral supplement increases the reproductive functions of breeding pigs, in particular, the number of stillborn piglets decreased by 1.64–7.70 % and their safety to weaning increased by 4.8–11.9 %. The increase of redox and metabolic processes in the animal body of the experimental groups positively affected the milking capacity of the breeding stock increasing it by 6.8–21.9 % and the growth of piglets, which was confirmed by their larger body weight by 3.3 and 4.6 %. The sorption properties of marl allowed reducing the concentration of toxic lead in the body of breeding pigs (by 40.17 and 42.01 %) and cadmium (by 20.57 and 24.42 %), decreasing the transition of cesium-137 isotope to beestings milk by 1.34 and 1.28 times. In areas with high soil pollution with radiocesium in Bryansk region, the use of natural marl sorbent in breeding pigs’ diets activates their immunity, hematopoietic functions, accelerates assimilation of protein and mineral exchange

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

Structural plasticity and thermal stability of the histone-like protein from <i>Spiroplasma melliferum</i> are due to phenylalanine insertions into the conservative scaffold

<p>The histone-like (HU) protein is one of the major nucleoid-associated proteins of the bacterial nucleoid, which shares high sequence and structural similarity with IHF but differs from the latter in DNA-specificity. Here, we perform an analysis of structural-dynamic properties of HU protein from <i>Spiroplasma melliferum</i> and compare its behavior in solution to that of another mycoplasmal HU from <i>Mycoplasma gallisepticum</i>. The high-resolution heteronuclear NMR spectroscopy was coupled with molecular-dynamics study and comparative analysis of thermal denaturation of both mycoplasmal HU proteins. We suggest that stacking interactions in two aromatic clusters in the HUSpm dimeric interface determine not only high thermal stability of the protein, but also its structural plasticity experimentally observed as slow conformational exchange. One of these two centers of stacking interactions is highly conserved among the known HU and IHF proteins. Second aromatic core described recently in IHFs and IHF-like proteins is considered as a discriminating feature of IHFs. We performed an electromobility shift assay to confirm high affinities of HUSpm to both normal and distorted dsDNA, which are the characteristics of HU protein. MD simulations of HUSpm with alanine mutations of the residues forming the non-conserved aromatic cluster demonstrate its role in dimer stabilization, as both partial and complete distortion of the cluster enhances local flexibility of HUSpm.</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

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

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>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

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