Conjugation with the Carrier Helped to Reveal acidification-Induced Structural Shift in the Peptide from Phospholipase Domain of Parvovirus B19.

Spectroscopic studies on domains and peptides of large proteins are complicated because of the tendency of short peptides to form oligomers in aquatic buffers, but conjugation of a peptide with a carrier protein may be helpful. In this study we approved that a fragment of SK30 peptide from phospholipase A2 domain of VP1 Parvovirus B19 capsid protein (residues: 144-159; 164; 171-183; sequence: SAVDSAARIHDFRYSQLAKLGINPYTHWTVADEELLKNIK) turns from random coil to alpha helix in the acidic medium only in case if it had been conjugated with BSA (through additional N-terminal Cys residue, turning it into CSK31 peptide, and SMCC linker) according to CD-spectroscopy results. In contrast, unconjugated SK30 peptide does not undergo such shift because it forms stable oligomers connected by intermolecular antiparallel beta sheet, according to IR-spectroscopy, CD-spectroscopy, blue native gel electrophoresis and centrifugal ultrafiltration, as, probably, the whole isolated phospholipase domain of VP1 protein does. However, being a part of the long VP1 capsid protein, phospholipase domain may change its fold during the acidification of the medium in the endolysosome by the way of the formation of contacts between protonated His153 and Asp175, promoting the shift from random coil to alpha helix in its N-terminal part. This study opens up a perspective of vaccine development, since rabbit polyclonal antibodies against the conjugate of CSK31 peptide with BSA, in which the structure of the second alpha helix from the phospholipase A2 domain should be reproduced, can bind epitopes of the complete recombinant unique part of VP1 Parvovirus B19 capsid (residues: 1-227).

Structural Shifts of the Parvovirus B19 Capsid Receptor-binding Domain: A Peptide Study.

BACKGROUND: Binding appropriate cellular receptors is a crucial step of a lifecycle for any virus. Structure of receptor-binding domain for a viral surface protein has to be determined before the start of future drug design projects. OBJECTIVE: Investigation of pH-induced changes in the secondary structure for a capsid peptide with loss of function mutation can shed some light on the mechanism of entrance. METHODS: Spectroscopic methods were accompanied by electrophoresis, ultrafiltration, and computational biochemistry. RESULTS: In this study, we showed that a peptide from the receptor-binding domain of Parvovirus B19 VP1 capsid (residues 13-31) is beta-structural at pH=7.4 in 0.01 M phosphate buffer, but alpha- helical at pH=5.0, according to the circular dichroism (CD) spectroscopy results. Results of infra- red (IR) spectroscopy showed that the same peptide exists in both alpha-helical and beta-structural conformations in partial dehydration conditions both at pH=7.4 and pH=5.0. In contrast, the peptide with Y20W mutation, which is known to block the internalization of the virus, forms mostly alpha-helical conformation in partial dehydration conditions at pH=7.4. According to our hypothesis, an intermolecular antiparallel beta structure formed by the wild-type peptide in its tetramers at pH=7.4 is the prototype of the similar intermolecular antiparallel beta structure formed by the corresponding part of Parvovirus B19 receptor-binding domain with its cellular receptor (AXL). CONCLUSION: Loss of function Y20W substitution in VP1 capsid protein prevents the shift into the beta-structural state by way of alpha helix stabilization and the decrease of its ability to turn into the disordered state.

Germline mutations directions are different between introns of the same gene: case study of the gene coding for amyloid-beta precursor protein.

Amyloid-beta precursor protein (APP) is highly conserved in mammals. This feature allowed us to compare nucleotide usage biases in fourfold degenerated sites along the length of its coding region for 146 species of mammals and birds in search of fragments with significant deviations. Even though cytosine usage has the highest value in fourfold degenerated sites in APP coding region from all tested placental mammals, in contrast to marsupial mammals with the bias toward thymine usage, the most frequent germline and somatic mutations in human APP coding region are C to T and G to A transitions. The same mutational AT-pressure is characteristic for germline mutations in introns of human APP gene. However, surprisingly, there are several exceptional introns with deviations in germline mutations rates. The most of those introns surround exons with exceptional biases in nucleotide usage in fourfold degenerated sites. Existence of such fragments in exons 4 and 5, as well as in exon 14, can be connected with the presence of lncRNA genes in complementary strand of DNA. Exceptional nucleotide usage bias in exons 16 and 17 that contain a sequence encoding amyloid-beta peptides can be explained either by the presence of yet unmapped lncRNA(s), or by the autonomous expression of a short mRNA that encodes just C-terminal part of the APP providing an alternative source of amyloid-beta peptides. This hypothesis is supported by the increased rate of T to C transitions in introns 16-17 and 17-18 of Human APP gene relatively to other introns.

Mechanism of curaxin-dependent nucleosome unfolding by FACT.

Human FACT (FACT) is a multifunctional histone chaperone involved in transcription, replication and DNA repair. Curaxins are anticancer compounds that induce FACT-dependent nucleosome unfolding and trapping of FACT in the chromatin of cancer cells (c-trapping) through an unknown molecular mechanism. Here, we analyzed the effects of curaxin CBL0137 on nucleosome unfolding by FACT using spFRET and electron microscopy. By itself, FACT adopted multiple conformations, including a novel, compact, four-domain state in which the previously unresolved NTD of the SPT16 subunit of FACT was localized, apparently stabilizing a compact configuration. Multiple, primarily open conformations of FACT-nucleosome complexes were observed during curaxin-supported nucleosome unfolding. The obtained models of intermediates suggest "decision points" in the unfolding/folding pathway where FACT can either promote disassembly or assembly of nucleosomes, with the outcome possibly being influenced by additional factors. The data suggest novel mechanisms of nucleosome unfolding by FACT and c-trapping by curaxins.

Diaminopelargonic acid transaminase from Psychrobacter cryohalolentis is active towards (S)-(-)-1-phenylethylamine, aldehydes and α-diketones.

Substrate and reaction promiscuity is a remarkable property of some enzymes and facilitates the adaptation to new metabolic demands in the evolutionary process. Substrate promiscuity is also a basis for protein engineering for biocatalysis. However, molecular principles of enzyme promiscuity are not well understood. Even for the widely studied PLP-dependent transaminases of class III, the reliable prediction of the biocatalytically important amine transaminase activity is still difficult if the desired activity is unrelated to the natural activity. Here, we show that 7,8-diaminopelargonic acid transaminase (synthase), previously considered to be highly specific, is able to convert (S)-(-)-1-phenylethylamine and a number of aldehydes and diketones. We were able to characterize the (S)-amine transaminase activity of 7,8-diaminopelargonic acid transaminase from Psychrobacter cryohalolentis (Pcryo361) and analyzed the three-dimensional structure of the enzyme. New substrate specificity for α-diketones was observed, though only a weak activity towards pyruvate was found. We examined the organization of the active site and binding modes of S-adenosyl-L-methionine and (S)-(-)-1-phenylethylamine using X-ray analysis and molecular docking. We suggest that the Pcryo361 affinity towards (S)-(-)-1-phenylethylamine arises from the recognition of the hydrophobic parts of the specific substrates, S-adenosyl-L-methionine and 7-keto-8-aminopelargonic acid, and from the flexibility of the active site. Our results support the observation that the conversion of amines is a promiscuous activity of many transaminases of class III and is independent from their natural function. The analysis of amine transaminase activity from among various transaminases will help to make the sequence-function prediction for biocatalysis more reliable.

Structural adaptations of octaheme nitrite reductases from haloalkaliphilic Thioalkalivibrio bacteria to alkaline pH and high salinity.

Bacteria Tv. nitratireducens and Tv. paradoxus from soda lakes grow optimally in sodium carbonate/NaCl brines at pH range from 9.5 to 10 and salinity from 0.5 to 1.5 M Na+. Octaheme nitrite reductases (ONRs) from haloalkaliphilic bacteria of genus Thioalkalivibrio are stable and active in a wide range of pH (up to 11) and salinity (up to 1 M NaCl). To establish adaptation mechanisms of ONRs from haloalkaliphilic bacteria a comparative analysis of amino acid sequences and structures of ONRs from haloalkaliphilic bacteria and their homologues from non-halophilic neutrophilic bacteria was performed. The following adaptation strategies were observed: (1) strategies specific for halophilic and alkaliphilic proteins (an increase in the number of aspartate and glutamate residues and a decrease in the number of lysine residues on the protein surface), (2) strategies specific for halophilic proteins (an increase in the arginine content and a decrease in the number of hydrophobic residues on the solvent-accessible protein surface), (3) strategies specific for alkaliphilic proteins (an increase in the area of intersubunit hydrophobic contacts). Unique adaptation mechanism inherent in the ONRs from bacteria of genus Thioalkalivibrio was revealed (an increase in the core in the number of tryptophan and phenylalanine residues, and an increase in the number of small side chain residues, such as alanine and valine, in the core).

Intramolecular hydrogen bonding in the polyextremophilic short-chain dehydrogenase from the archaeon Thermococcus sibiricus and its close structural homologs.

The short-chain alcohol dehydrogenase from the archaeon Thermococcus sibiricus (TsAdh319) exhibits adaptation to different kinds of stress: high temperature, high salinity, and the presence of organic solvents and denaturants. Previously a comparison of TsAdh319 with close structural homologs revealed an abnormally large number of charged residues on the surface of TsAdh319 tetramer. We further focused on the analysis of hydrogen bonding of TsAdh319 and its structural homologs from thermophilic and mesophilic organisms as a structural factor of adaptation to extreme environment. The calculation and analysis of the dynamics of hydrogen bonds of different kind were performed. In particular, the intramolecular hydrogen bonds of different kind according to their location and the type of a.a. residues involved in the bond were analyzed. TsAdh319 showed the greatest contribution of charged residues to the formation of surface hydrogen bonds, inner hydrogen bonding, and the bonds between different subunits compared to its structural homologs. Molecular dynamics simulations revealed that, of three enzyme molecules analyzed, TsAdh319 shows the least change in the number of hydrogen bonds of different kinds upon a temperature shift from 27 to 85 °C. The greatest changes were observed for a homologous enzyme from a mesophilic host. Only guanidine hydrochloride being a charged agent was able to deactivate TsAdh319. We suggest that the percentage of charged residues plays a key role in the resistance of TsAdh319 to environmental stress. The analysis shows that salt bridges in TsAdh319 serve as a universal instrument of stabilization under different extreme conditions.

Domain structure and conformational changes in rat KV2.1 ion channel.

Voltage-gated potassium Kv2.1 channels are widely distributed in the central nervous system, specifically in neuroendocrine and endocrine cells. Their cytoplasmic C-termini are large and carry out many important functions. Here we provide the first direct structural evidence that each C-terminal part within the Kv2.1 ion channel is formed by two distinct domains (Kv2 and CTA). We expressed and purified two C-terminal truncation mutants of a rat Kv2.1 channel, lacking the entire C-termini or the CTA domain. Single particle electron microscopy was used to obtain three-dimensional reconstructions of purified C-terminal Kv2.1 mutants at 2.0 and 2.4 nm resolution. Comparison of these structures to each other and to the low-resolution EM structure of the full-length Kv2.1 channel revealed the exact locations of cytoplasmic Kv2 and CTA domains within the tetramer. Four Kv2 domains envelop the N-terminal T1 domain. The tetramer of the CTA domains underlies the Kv2-T1 complex and may also affect the channel's surface expression. Subsequent molecular dynamics simulation and homology modeling produced open and closed structural models of the membrane part of the Kv2.1 channel.