A Novel Two-Domain Laccase with Middle Redox Potential: Physicochemical and Structural Properties.

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.

Structural Insight into the Amino Acid Environment of the Two-Domain Laccase's Trinuclear Copper Cluster.

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.

The RNA-Binding and RNA-Melting Activities of the Multifunctional Protein Nucleobindin 1.

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.

Two Epitope Regions Revealed in the Complex of IL-17A and Anti-IL-17A VHH Domain.

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 ß-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.

Structural and Functional Characterization of ß-lytic Protease from Lysobacter capsici VKM B-2533T.

The crystal structure of the Lysobacter capsici VKM B-2533T ß-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.

Differential Impact of Hexuronate Regulators ExuR and UxuR on the Escherichia coli Proteome.

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.

The role of positive charged residue in the proton-transfer mechanism of two-domain laccase from Streptomyces griseoflavus Ac-993.

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.

Structure and RNA-Binding Properties of Lsm Protein from Halobacterium salinarum.

The structure and the RNA-binding properties of the Lsm protein from Halobacterium salinarum have been determined. A distinctive feature of this protein is the presence of a short L4 loop connecting the ß3 and ß4 strands. Since bacterial Lsm proteins (also called Hfq proteins) have a short L4 loop and form hexamers, whereas archaeal Lsm proteins (SmAP) have a long L4 loop and form heptamers, it has been suggested that the length of the L4 loop may affect the quaternary structure of Lsm proteins. Moreover, the L4 loop covers the region of SmAP corresponding to one of the RNA-binding sites in Hfq, and thus can affect the RNA-binding properties of the protein. Our results show that the SmAP from H. salinarum forms heptamers and possesses the same RNA-binding properties as homologous proteins with the long L4 loop. Therefore, the length of the L4 does not govern the number of monomers in the protein particles and does not affect the RNA-binding properties of Lsm proteins.

Characterization of Regulatory Elements of L11 and L1 Operons in Thermophilic Bacteria and Archaea.

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.

Engineering the Catalytic Properties of Two-Domain Laccase from Streptomyces griseoflavus Ac-993.

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.

Dimerization of long hibernation promoting factor from Staphylococcus aureus: Structural analysis and biochemical characterization.

Staphylococcus aureus hibernation promoting factor (SaHPF) is responsible for the formation of 100S ribosome dimers, which in turn help this pathogen to reduce energy spent under unfavorable conditions. Ribosome dimer formation strongly depends on the dimerization of the C-terminal domain of SaHPF (CTDSaHPF). In this study, we solved the crystal structure of CTDSaHPF at 1.6 Šresolution and obtained a precise arrangement of the dimer interface. Residues Phe160, Val162, Thr171, Ile173, Tyr175, Ile185 andThr187 in the dimer interface of SaHPF protein were mutated and the effects were analyzed for the formation of 100S disomes of ribosomes isolated from S. aureus. It was shown that substitution of any of single residues Phe160, Val162, Ile173, Tyr175 and Ile185 in the SaHPF homodimer interface abolished the ribosome dimerization in vitro.

Investigations of Accessibility of T2/T3 Copper Center of Two-Domain Laccase from Streptomyces griseoflavus Ac-993.

Laccases (EC 1.10.3.2) are multicopper oxidoreductases acting on diphenols and related substances. Laccases are highly important for biotechnology and environmental remediation. These enzymes contain mononuclear one T2 copper ion and two T3 copper ions (Cu3α and Cu3ß), which form the so-called trinuclear center (TNC). Along with the typical three-domain laccases Bacteria produce two-domain (2D) enzymes, which are active at neutral and basic pH, thermostable, and resistant to inhibitors. In this work we present the comparative analysis of crystal structures and catalytic properties of recombinant 2D laccase from Streptomyces griseoflavus Ac-993 (SgfSL) and its four mutant forms with replacements of two amino acid residues, located at the narrowing of the presumable T3-solvent tunnels. We obtained inactive enzymes with substitutions of His165, with Phe, and Ile170 with Ala or Phe. His165Ala variant was more active than the wild type. We suggest that His165 is a "gateway" at the O2-tunnel leading from solvent to the Cu3ß of the enzyme. The side chain of Ile170 could be indirectly involved in the coordination of copper ions at the T3 center by maintaining the position of the imidazole ring of His157 that belongs to the first coordination sphere of Cu3α.

An Experimental Tool to Estimate the Probability of a Nucleotide Presence in the Crystal Structures of the Nucleotide-Protein Complexes.

A correlation between the ligand-protein affinity and the identification of the ligand in the experimental electron density maps obtained by X-ray crystallography has been tested for a number of RNA-binding proteins. Bacterial translation regulators ProQ, TRAP, Rop, and Hfq together with their archaeal homologues SmAP have been used. The equilibrium dissociation constants for the N-methyl-anthraniloyl-labelled adenosine and guanosine monophosphates titrated by the proteins have been determined by the fluorescent anisotropy measurements. The estimated stability of the nucleotide-protein complexes has been matched with a presence of the nucleotides in the structures of the proposed nucleotide-protein complexes. It has been shown that the ribonucleotides can be definitely identified in the experimental electron density maps at equilibrium dissociation constant <10 µM. At KD of 20-40 µM, long incubation of the protein crystals in the nucleotide solution is required to obtain the structures of the complexes. The complexes with KD value higher than 50 µM are not stable enough to survive in crystallization conditions.

Characterization of RNA-binding properties of the archaeal Hfq-like protein from Methanococcus jannaschii.

The Sm and Sm-like proteins are widely distributed among bacteria, archaea and eukarya. They participate in many processes related to RNA-processing and regulation of gene expression. While the function of the bacterial Lsm protein Hfq and eukaryotic Sm/Lsm proteins is rather well studied, the role of Lsm proteins in Archaea is investigated poorly. In this work, the RNA-binding ability of an archaeal Hfq-like protein from Methanococcus jannaschii has been studied by X-ray crystallography, anisotropy fluorescence and surface plasmon resonance. It has been found that MjaHfq preserves the proximal RNA-binding site that usually recognizes uridine-rich sequences. Distal adenine-binding and lateral RNA-binding sites show considerable structural changes as compared to bacterial Hfq. MjaHfq did not bind mononucleotides at these sites and would not recognize single-stranded RNA as its bacterial homologues. Nevertheless, MjaHfq possesses affinity to poly(A) RNA that seems to bind at the unstructured positive-charged N-terminal tail of the protein.

Structural Studies of Component of Lysoamidase Bacteriolytic Complex from Lysobacter sp. XL1.

The lysoamidase bacteriolytic complex (LBC) comprising five enzymes (L1-L5) is secreted into the culture liquid by gram-negative bacterium Lysobacter sp. XL1. The medicinal agent lysoamidase has a broad-antimicrobial spectrum. Bacteriolytic protease L1 belongs to the LBC. Recombinant L1 protease of Lysobacter sp. XL1 was expressed, purified to homogeneity and crystallized. The X-ray structure of L1 at 1.35 Å resolution has been determined using the synchrotron data and the molecular replacement method. L1 protease is a thermostable whose thermal unfolding proceeds in one step without forming stable intermediates. Structural information concerning L1 will contribute to the development of new-generation antimicrobial drugs, whose application will not be accompanied by the selection of resistant microorganisms.

Crystallization and X-ray diffraction studies of a two-domain laccase from Streptomyces griseoflavus.

Laccase (EC 1.10.3.2) is one of the most common copper-containing oxidases; it is found in many organisms and catalyzes the oxidation of primarily phenolic compounds by oxygen. Two-domain laccases have unusual thermostability, resistance to inhibitors and an alkaline optimum of activity. The causes of these properties in two-domain laccases are poorly understood. A recombinant two-domain laccase (SgfSL) was cloned from the genome of Streptomyces griseoflavus Ac-993, expressed in Escherichia coli and purified to homogeneity. The crystals of SgfSL belonged to the monoclinic space group P21, with unit-cell parameters a = 74.64, b = 94.72, c = 117.40 Å, ß = 90.672°, and diffraction data were collected to 2.0 Šresolution using a synchrotron-radiation source. Two functional trimers per asymmetric unit correspond to a Matthews coefficient of 1.99 Å(3) Da(-1) according to the monomer molecular weight of 35.6 kDa.

The Activation Mechanism of 2'-5'-Oligoadenylate Synthetase Gives New Insights Into OAS/cGAS Triggers of Innate Immunity.

2'-5'-Oligoadenylate synthetases (OASs) produce the second messenger 2'-5'-oligoadenylate, which activates RNase L to induce an intrinsic antiviral state. We report on the crystal structures of catalytic intermediates of OAS1 including the OAS1·dsRNA complex without substrates, with a donor substrate, and with both donor and acceptor substrates. Combined with kinetic studies of point mutants and the previously published structure of the apo form of OAS1, the new data suggest a sequential mechanism of OAS activation and show the individual roles of each component. They reveal a dsRNA-mediated push-pull effect responsible for large conformational changes in OAS1, the catalytic role of the active site Mg(2+), and the structural basis for the 2'-specificity of product formation. Our data reveal similarities and differences in the activation mechanisms of members of the OAS/cyclic GMP-AMP synthase family of innate immune sensors. In particular, they show how helix 3103-α5 blocks the synthesis of cyclic dinucleotides by OAS1.

Protein-RNA affinity of ribosomal protein L1 mutants does not correlate with the number of intermolecular interactions.

Ribosomal protein L1, as part of the L1 stalk of the 50S ribosomal subunit, is implicated in directing tRNA movement through the ribosome during translocation. High-resolution crystal structures of four mutants (T217V, T217A, M218L and G219V) of the ribosomal protein L1 from Thermus thermophilus (TthL1) in complex with a specific 80 nt fragment of 23S rRNA and the structures of two of these mutants (T217V and G219V) in the RNA-unbound form are reported in this work. All mutations are located in the highly conserved triad Thr-Met-Gly, which is responsible for about 17% of all protein-RNA hydrogen bonds and 50% of solvent-inaccessible intermolecular hydrogen bonds. In the mutated proteins without bound RNA the RNA-binding regions show substantial conformational changes. On the other hand, in the complexes with RNA the structures of the RNA-binding surfaces in all studied mutants are very similar to the structure of the wild-type protein in complex with RNA. This shows that formation of the RNA complexes restores the distorted surfaces of the mutant proteins to a conformation characteristic of the wild-type protein complex. Domain I of the mutated TthL1 and helix 77 of 23S rRNA form a rigid body identical to that found in the complex of wild-type TthL1 with RNA, suggesting that the observed relative orientation is conserved and is probably important for ribosome function. Analysis of the complex structures and the kinetic data show that the number of intermolecular contacts and hydrogen bonds in the RNA-protein contact area does not correlate with the affinity of the protein for RNA and cannot be used as a measure of affinity.

Studying the properties of domain I of the ribosomal protein l1: incorporation into ribosome and regulation of the l1 operon expression.

L1 is a conserved protein of the large ribosomal subunit. This protein binds strongly to the specific region of the high molecular weight rRNA of the large ribosomal subunit, thus forming a conserved flexible structural element--the L1 stalk. L1 protein also regulates translation of the operon that comprises its own gene. Crystallographic data suggest that L1 interacts with RNA mainly by means of its domain I. We show here for the first time that the isolated domain I of the bacterial protein L1 of Thermus thermophilus and Escherichia coli is able to incorporate in vivo into the E. coli ribosome. Furthermore, domain I of T. thermophilus L1 can regulate expression of the L1 gene operon of Archaea in the coupled transcription-translation system in vitro, as well as the intact protein. We have identified the structural elements of domain I of the L1 protein that may be responsible for its regulatory properties.

Double suppression of the Gα protein activity by RGS proteins.

Regulator of G protein signaling (RGS) proteins accelerate GTP hydrolysis on G protein α subunits, restricting their activity downstream from G protein-coupled receptors. Here we identify Drosophila Double hit (Dhit) as a dual RGS regulator of Gαo. In addition to the conventional GTPase-activating action, Dhit possesses the guanine nucleotide dissociation inhibitor (GDI) activity, slowing the rate of GTP uptake by Gαo; both activities are mediated by the same RGS domain. These findings are recapitulated using homologous mammalian Gαo/i proteins and RGS19. Crystal structure and mutagenesis studies provide clues into the molecular mechanism for this unprecedented GDI activity. Physiologically, we confirm this activity in Drosophila asymmetric cell divisions and HEK293T cells. We show that the oncogenic Gαo mutant found in breast cancer escapes this GDI regulation. Our studies identify Dhit and its homologs as double-action regulators, inhibiting Gαo/i proteins both through suppression of their activation and acceleration of their inactivation through the single RGS domain.