Representatives of the colorless sulfur bacteria of the genus Beggiatoa use reduced sulfur compounds in the processes of lithotrophic growth, which is accompanied by the storage of intracellular sulfur. However, it is still unknown how the transformation of intracellular sulfur occurs in Beggiatoa representatives. Annotation of the genome of Beggiatoa leptomitoformis D-402 did not identify any genes for the oxidation or reduction of elemental sulfur. By searching BLASTP, two putative persulfide dioxygenase (PDO) homologs were found in the genome of B. leptomitoformis. In some heterotrophic prokaryotes, PDO is involved in the oxidation of sulfane sulfur. According to HPLC-MS/MS, the revealed protein was reliably detected in a culture sample grown only in the presence of endogenous sulfur and CO2. The recombinant protein from B. leptomitoformis was active in the presence of glutathione persulfide. The crystal structure of recombinant PDO exhibited consistency with known structures of type I PDO. Thus, it was shown that B. leptomitoformis uses PDO to oxidize endogenous sulfur. Additionally, on the basis of HPLC-MS/MS, RT-qPCR, and the study of PDO reaction products, we predicted the interrelation of PDO and Sox-system function in the oxidation of endogenous sulfur in B. leptomitoformis and the connection of this process with energy metabolism.

The search for new biomarkers for the early diagnosis of systemic lupus erythematosus (SLE) is a crucial task. Objective: a comparative study of concentrations of conservative protein nucleobindin 1 (NUCB1) in the blood serum of patients with SLE and healthy donors and assessment of correlation of NUCB1 level with clinical and serological manifestations of the disease.Material and methods. The study included 21 patients with SLE who fulfilled SLICC criteria and 23 healthy donors. SLEDAI-2K index was used to assess SLE activity. Organ damage was assessed using SLICC damage index. Standard laboratory markers of SLE were analyzed in all patients. Concentration of NUCB1 in blood serum was determined using the enzyme immunoassay method. Results and discussion. The group of SLE patients included 20 women and 1 man (median age 33 [27; 40] years, disease duration 5 [3; 10] years), mainly with high disease activity (median SLEDAI-2K 8.5 [6.0; 14.0]). Kidney involvement was found in 52% of cases (nephritis), involvement of joints – in 67% (arthritis), vessels – in 33%, skin – in 67%, pericarditis – in 29%, hematological abnormalities – in 71%, antinuclear factor – in 76% and antibodies against double-stranded DNA – in 71%. An increase in the mean NUCB1 level to 3881 ng/ml was found in the blood serum of SLE patients compared to the control group (2766 ng/ml; p=0.048). Correlations of NUCB1 levels with vascular damage (r=0.653; p<0.05) and pericarditis (r=-0.490; p<0.05) were found. Conclusion. Elevated NUCB1 levels in the blood serum of SLE patients may indicate involvement of this protein in autoimmune and apoptotic processes. The observed correlation of NUCB1 levels with vascular affection and pericarditis is the basis for further studies on the involvement of this protein in the development of various diseases, including SLE.

Analysis of the structure of two-domain laccase ScaSL from Streptomyces carpinensis VKM Ac-1300 (with a middle-redox potential) revealed determinants that could affect the increased potential of ScaSL. Site-directed mutagenesis of the ScaSL laccase was carried out, and mutants H286A, H286T, H286W, and F232Y/F233Y were obtained. Replacement of His 286 with Ala led to a decrease in redox potential (0.45 V) and an increase in stability at pH 9 and 11; replacement with Thr led to an increase in redox potential (0.51 V) but to a decrease in the thermal stability of the protein; replacement with Trp did not affect the enzyme properties. Replacement of Phe residues 232 and 233 with Tyr led to a shift in enzyme activity to the acidic pH range without changing the redox potential and a decrease in the thermostability and pH stability of the enzyme. All mutants more efficiently oxidized phenolic substrate 2,6-DMP and were able to participate in direct electron transfer (DET) with MWCNT-modified electrodes. The F232Y/F233/Y mutant was unable to degrade triphenylmethane dyes without a mediator but showed a greater degree of decolorization of azo dyes in the presence of the mediator. The crystal structure of laccase with the highest potential was determined with high resolution.

The gene for a previously unexplored two-domain laccase was identified in the genome of actinobacterium Streptomyces carpinensis VKM Ac-1300. The two-domain laccase, named ScaSL, was produced in a heterologous expression system (Escherichia coli strain M15 [pREP4]). The enzyme was purified to homogeneity using affinity chromatography. ScaSL laccase, like most two-domain laccases, exhibited activity in the homotrimer form. However, unlike the most two-domain laccases, it was also active in multimeric forms. The enzyme exhibited maximum activity at 80°C and was thermally stable. Half-inactivation time of ScaSL at 80°C was 40 min. The laccase was able to oxidize a non-phenolic organic compound ABTS at a maximum rate at pH 4.7, and to oxidized a phenolic compound 2,6-dimethoxyphenol at a maximum rate at pH 7.5. The laccase stability was observed in the pH range 9-11. At pH 7.5, laccase was slightly inhibited by sodium azide, sodium fluoride, and sodium chloride; at pH 4.5, the laccase was completely inhibited by 100 mM sodium azide. The determined Km and kcat of the enzyme for ABTS were 0.1 mM and 20 s–1, respectively. The Km and kcat for 2,6-dimethoxyphenol were 0.84 mM and 0.36 s–1, respectively. ScaSL catalyzed polymerization of humic acids and lignin. Redox potential of the laccase was 0.472 ± 0.007 V. Thus, the ScaSL laccase is the first characterized two-domain laccase with a middle redox potential. Crystal structure of ScaSL was determined with 2.35 Å resolution. Comparative analysis of the structures of ScaSL and other two-domain laccases suggested that the middle potential of ScaSL may be associated with conformational differences in the position of the side groups of amino acids at position 230 (in ScaSL numbering), which belong to the second coordination sphere of the copper atom of the T1 center.

Nucleobindin 1 (NUCB1) is a ubiquitous multidomain protein that belongs to the EF-hand Ca2+-binding superfamily. NUCB1 interacts with Galphai3 protein, cyclooxygenase, amyloid precursor protein, and lipids. It is involved in stress response and human diseases. In addition, this protein is a transcription factor that binds to the DNA E-box motif. Using surface plasmon resonance and molecular beacon approaches, we first showed the RNA binding and RNA melting activities of NUCB1. We suggest that NUCB1 could induce local changes in structured RNAs via binding to the GGAUAU loop sequence. Our results demonstrate the importance of the multidomain structure of NUCB1 for its RNA-chaperone activity in vitro.

Laccases are industrially relevant enzymes. However, their range of applications is limited by their functioning and stability. Most of the currently known laccases function in acidic conditions at temperatures below 60 °C, but two-domain laccases (2D) oxidize some substrates in alkaline conditions and above 70 °C. In this study, we aim to establish the structural factors affecting the alkaline activity of the 2D laccase from Streptomyces griseoflavus (SgfSL). The range of methods used allowed us to show that the alkaline activity of SgfSL is influenced by the polar residues located close to the trinuclear center (TNC). Structural and functional studies of the SgfSL mutants Met199Ala/Asp268Asn and Met199Gly/Asp268Asn revealed that the substitution Asp268Asn (11 Å from the TNC) affects the orientation of the Asn261 (the second coordination sphere of the TNC), resulting in hydrogen-bond-network reorganization, which leads to a change in the SgfSL-activity pH profile. The combination of the Met199Gly/Arg240His and Asp268Asn substitutions increased the efficiency (kcat/KM) of the 2,6-DMP oxidation by 34-fold compared with the SgfSL. Our results extend the knowledge about the structure and functioning of 2D laccases' TNC active sites and open up new possibilities for the directed engineering of laccases.

Multi-copper oxidases are capable of coupling the one-electron oxidation of four substrate equivalents to the four-electron reduction of dioxygen to two molecules of water. This process takes place at the trinuclear copper center of the enzymes. Previously, the main catalytic stages for three-domain (3D) laccases have been identified. However, for bacterial small two-domain (2D) laccases several questions remain to be answered. One of them is the nature of the protonation events upon the reductive cleavage of dioxygen to water. In 3D laccases, acidic residues play a key role in the protonation mechanisms. In this study, the role of the Arg240 residue, located within the T2 tunnel of 2D laccase from Streptomyces griseoflavus Ac-993, was investigated. X-ray structural analysis and kinetic characterization of two mutants, R240A and R240H, have provided support for a role of this residue in the protonation events. Communicated by Ramaswamy H. Sarma

The crystal structure of the Lysobacter capsici VKM B−2533T β-lytic protease (Blp), a medicinally promising antimicrobial enzyme, was first solved. Blp was established to possess a folding characteristic of the M23 protease family. The groove of the Blp active site, as compared with that of the LasA structural homologue from Pseudomonas aeruginosa, was found to have amino acid differences. Biochemical analysis revealed no differences in the optimal reaction conditions for manifesting Blp and LasA bacteriolytic activities. At the same time, Blp had a broader range of action against living and autoclaved target cells. The results suggest that the distinction in the geometry of the active site and the charge of amino acid residues that form the active site groove can be important for the hydrolysis of different peptidoglycan types in target cells.

ExuR and UxuR are paralogous proteins belonging to the GntR family of transcriptional regulators. Both are known to control hexuronic acid metabolism in a variety of Gammaproteobacteria but the relative impact of each of them is still unclear. Here, we apply 2D difference electrophoresis followed by mass-spectrometry to characterise the changes in the Escherichia coli proteome in response to a uxuR or exuR deletion. Our data clearly show that the effects are different: deletion of uxuR resulted in strongly enhanced expression of D-mannonate dehydratase UxuA and flagellar protein FliC, and in a reduced amount of outer membrane porin OmpF, while the absence of ExuR did not significantly alter the spectrum of detected proteins. Consequently, the physiological roles of proteins predicted as homologs seem to be far from identical. Effects of uxuR deletion were largely dependent on the cultivation conditions: during growth with glucose, UxuA and FliC were dramatically altered, while during growth with glucuronate, activation of both was not so prominent. During the growth with glucose, maximal activation was detected for FliC. This was further confirmed by expression analysis and physiological tests, thus suggesting the involvement of UxuR in the regulation of bacterial motility and biofilm formation.

Laccases catalyze the oxidation of substrates with the concomitant reduction of oxygen to water. Recently, we found that polar residues located in tunnels leading to Cu2 and Cu3 ions control oxygen entrance (His 165) and proton transport (Arg 240) of two-domain laccase (2D) from Streptomyces griseoflavus (SgfSL). In this work, we have focused on optimizing the substrate-binding pocket (SBP) of SgfSL while simultaneously adjusting the oxygen reduction process. SgfSL variants with three single (Met199Ala, Met199Gly, and Tyr230Ala) and three double amino acid residues substitutions (Met199Gly/His165Ala, His165Ala/Arg240His, Met199Gly/Arg240His) were constructed, purified, and investigated. Combination of substitutions in the SBP and in the tunnel leading to Cu2 ion (Met199Gly/Arg240His) increased SgfSL catalytic activity towards ABTS by 5-fold, and towards 2.6-DMP by 16-fold. The high activity of the Met199Gly/Arg240His variant can be explained by the combined effect of the SBP geometry optimization (Met199Gly) and increased proton flux via the tunnel leading to Cu2 ion (Arg240His). Moreover, the variant with Met199Gly and His165Ala mutations did not significantly increase SgfSL’s activity, but led to a drastic shift in the optimal pH of 2.6-DMP oxidation. These results indicate that His 165 not only regulates oxygen access, but it also participates in proton transport in 2D laccases.

Structural studies of RNA–protein complexes are important for understanding many molecular mechanisms occurring in cells (e.g., regulation of protein synthesis and RNA-chaperone activity of proteins). Various objects investigated at the Institute of Protein Research of the Russian Academy of Sciences are considered. Based on the analysis of the structures of the complexes of the ribosomal protein L1 with specific regions on both mRNA and rRNA, the principles of regulation of the translation of the mRNA of its own operon are presented. The studies of the heterotrimeric translation initiation factor IF2 of archaea and eukaryotes are described, and the data on the interaction of glycyl-tRNA-synthetase with viral IRES are reported. The results of studying the interaction of RNA molecules with one of functionally important sites of the Hfq protein are presented, and the differences in the RNA-binding properties of the Hfq and archaeal Lsm proteins are revealed.

Interleukin-17 (IL-17) is a cytokine produced by the Th17 cells. It is involved in chronic inflammation in patients with autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, and psoriasis. The antibodies targeting IL-17 and/or IL-17R are therapy tools for these diseases. Netakimab is an IL-17A-specific antibody containing a Lama glama VHH derivative domain and a VL variable domain. We have determined the crystal structure of the IL-17A-specific VHH domain in complex with IL-17A at 2.85 Å resolution. Certain amino acid residues of the three complementary-determining regions of the VHH domain form a network of solvent-inaccessible hydrogen bonds with two epitope regions of IL-17A. The β-turn of IL-17A, which forms the so-called epitope-1, appears to be the main region of IL-17A interaction with the antibody. Contacts formed by the IL-17A mobile C-terminal region residues (epitope-2) further stabilize the antibody–antigen complex.

Ribosomal protein L1 is a conserved two-domain protein that is involved in formation of the L1 stalk of the large ribosomal subunit. When there are no free binding sites available on the ribosomal 23S RNA, the protein binds to the specific site on the mRNA of its own operon (L11 operon in bacteria and L1 operon in archaea) preventing translation. Here we show that the regulatory properties of the r-protein L1 and its domain I are conserved in the thermophilic bacteria Thermus and Thermotoga and in the halophilic archaeon Haloarcula marismortui. At the same time the revealed features of the operon regulation in thermophilic bacteria suggest presence of two regulatory regions.