Bright and stable monomeric green fluorescent protein derived from StayGold.

The high brightness and photostability of the green fluorescent protein StayGold make it a particularly attractive probe for long-term live-cell imaging; however, its dimeric nature precludes its application as a fluorescent tag for some proteins. Here, we report the development and crystal structures of a monomeric variant of StayGold, named mBaoJin, which preserves the beneficial properties of its precursor, while serving as a tag for structural proteins and membranes. Systematic benchmarking of mBaoJin against popular green fluorescent proteins and other recently introduced monomeric and pseudomonomeric derivatives of StayGold established mBaoJin as a bright and photostable fluorescent protein, exhibiting rapid maturation and high pH/chemical stability. mBaoJin was also demonstrated for super-resolution, long-term live-cell imaging and expansion microscopy. We further showed the applicability of mBaoJin for neuronal labeling in model organisms, including Caenorhabditis elegans and mice.

Bacteriophage T5 dUTPase: Combination of Common Enzymatic and Novel Functions.

The main function of dUTPases is to regulate the cellular levels of dUTP and dTTP, thereby playing a crucial role in DNA repair mechanisms. Despite the fact that mutant organisms with obliterated dUTPase enzymatic activity remain viable, it is not possible to completely knock out the dut gene due to the lethal consequences of such a mutation for the organism. As a result, it is considered that this class of enzymes performs an additional function that is essential for the organism's survival. In this study, we provide evidence that the dUTPase of bacteriophage T5 fulfills a supplemental function, in addition to its canonical role. We determined the crystal structure of bacteriophage T5 dUTPase with a resolution of 2.0 Å, and we discovered a distinct short loop consisting of six amino acid residues, representing a unique structural feature specific to the T5-like phages dUTPases. The removal of this element did not affect the overall structure of the homotrimer, but it had significant effects on the development of the phage. Furthermore, it was shown that the enzymatic function and the novel function of the bacteriophage T5 dUTPase are unrelated and independent from each other.

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.

Stabilization of Cereibacter sphaeroides Photosynthetic Reaction Center by the Introduction of Disulfide Bonds.

The photosynthetic reaction center of the purple nonsulfur bacterium Cereibacter sphaeroides is a useful model for the study of mechanisms of photoinduced electron transfer and a promising component for photo-bio-electrocatalytic systems. The basic research and technological applications of this membrane pigment-protein complex require effective approaches to increase its structural stability. In this work, a rational design approach to genetically modify the reaction centers by introducing disulfide bonds is used. This resulted in significantly increasing the thermal stability of some of the mutant pigment-protein complexes. The formation of the S-S bonds was confirmed by X-ray crystallography as well as SDS-PAGE, and the optical properties of the reaction centers were studied. The genetically modified reaction centers presented here preserved their ability for photochemical charge separation and could be of interest for basic science and biotechnology.

Properties and Crystal Structure of the Cereibacter sphaeroides Photosynthetic Reaction Center with Double Amino Acid Substitution I(L177)H + F(M197)H.

The photosynthetic reaction center of the purple bacterium Cereibacter sphaeroides with two site-directed mutations Ile-L177-His and M197 Phe-His is of double interest. The substitution I(L177)H results in strong binding of a bacteriochlorophyll molecule with L-subunit. The second mutation F(M197)H introduces a new H-bond between the C2-acetyl carbonyl group of the bacteriochlorophyll PB and His-M197, which is known to enhance the stability of the complex. Due to this H-bond, π -electron system of P finds itself connected to an extensive H-bonding network on the periplasmic surface of the complex. The crystal structure of the double mutant reaction center obtained with 2.6 Å resolution allows clarifying consequences of the Ile L177 - His substitution. The value of the P/P+ midpoint potential in the double mutant RC was found to be ~20 mV less than the sum of potentials measured in the two RCs with single mutations I(L177)H and F(M197)H. The protein environment of the BChls PA and BB were found to be similar to that in the RC with single substitution I(L177)H, whereas an altered pattern of the H-bonding networks was found in the vicinity of bacteriochlorophyll PB. The data obtained are consistent with our previous assumption on a correlation between the bulk of the H-bonding network connected with the π-electron system of the primary electron donor P and the value of its oxidation potential.

Yet Another Similarity between Mitochondrial and Bacterial Ribosomal Small Subunit Biogenesis Obtained by Structural Characterization of RbfA from S. aureus.

Ribosome biogenesis is a complex and highly accurate conservative process of ribosomal subunit maturation followed by association. Subunit maturation comprises sequential stages of ribosomal RNA and proteins' folding, modification and binding, with the involvement of numerous RNAses, helicases, GTPases, chaperones, RNA, protein-modifying enzymes, and assembly factors. One such assembly factor involved in bacterial 30S subunit maturation is ribosomal binding factor A (RbfA). In this study, we present the crystal (determined at 2.2 Å resolution) and NMR structures of RbfA as well as the 2.9 Å resolution cryo-EM reconstruction of the 30S-RbfA complex from Staphylococcus aureus (S. aureus). Additionally, we show that the manner of RbfA action on the small ribosomal subunit during its maturation is shared between bacteria and mitochondria. The obtained results clarify the function of RbfA in the 30S maturation process and its role in ribosome functioning in general. Furthermore, given that S. aureus is a serious human pathogen, this study provides an additional prospect to develop antimicrobials targeting bacterial pathogens.

Nonspecific Amyloid Aggregation of Chicken Smooth-Muscle Titin: In Vitro Investigations.

A giant multidomain protein of striated and smooth vertebrate muscles, titin, consists of tandems of immunoglobulin (Ig)- and fibronectin type III (FnIII)-like domains representing ß-sandwiches, as well as of disordered segments. Chicken smooth muscles express several titin isoforms of ~500-1500 kDa. Using various structural-analysis methods, we investigated in vitro nonspecific amyloid aggregation of the high-molecular-weight isoform of chicken smooth-muscle titin (SMTHMW, ~1500 kDa). As confirmed by X-ray diffraction analysis, under near-physiological conditions, the protein formed amorphous amyloid aggregates with a quaternary cross-ß structure within a relatively short time (~60 min). As shown by circular dichroism and Fourier-transform infrared spectroscopy, the quaternary cross-ß structure-unlike other amyloidogenic proteins-formed without changes in the SMTHMW secondary structure. SMTHMW aggregates partially disaggregated upon increasing the ionic strength above the physiological level. Based on the data obtained, it is not the complete protein but its particular domains/segments that are likely involved in the formation of intermolecular interactions during SMTHMW amyloid aggregation. The discovered properties of titin position this protein as an object of interest for studying amyloid aggregation in vitro and expanding our views of the fundamentals of amyloidogenesis.

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.

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.

Y98 Mutation Leads to the Loss of RsfS Anti-Association Activity in Staphylococcus aureus.

Ribosomal silencing factor S (RsfS) is a conserved protein that plays a role in the mechanisms of ribosome shutdown and cell survival during starvation. Recent studies demonstrated the involvement of RsfS in the biogenesis of the large ribosomal subunit. RsfS binds to the uL14 ribosomal protein on the large ribosomal subunit and prevents its association with the small subunit. Here, we estimated the contribution of RsfS amino acid side chains at the interface between RsfS and uL14 to RsfS anti-association function in Staphylococcus aureus through in vitro experiments: centrifugation in sucrose gradient profiles and an S. aureus cell-free system assay. The detected critical Y98 amino acid on the RsfS surface might become a new potential target for pharmacological drug development and treatment of S. aureus infections.

Is RsfS a Hibernation Factor or a Ribosome Biogenesis Factor?

Solving the structures of bacterial, archaeal, and eukaryotic ribosomes by crystallography and cryo-electron microscopy has given an impetus for studying intracellular regulatory proteins affecting various stages of protein translation. Among them are ribosome hibernation factors, which have been actively investigated during the last decade. These factors are involved in the regulation of protein biosynthesis under stressful conditions. The main role of hibernation factors is the reduction of energy consumption for protein biosynthesis and preservation of existing functional ribosomes from degradation, which increases cell survival under unfavorable conditions. Despite a broad interest in this topic, only a few articles have been published on the ribosomal silencing factor S (RsfS). According to the results of these studies, RsfS can be assigned to the group of hibernation factors. However, recent structural studies of the 50S ribosomal subunit maturation demonstrated that RsfS has the features inherent to biogenesis factors for example, ability to bind to the immature ribosomal subunit (similar to the RsfS mitochondrial ortholog MALSU1, mitochondrial assembly of ribosomal large subunit 1). In this review, we summarized the information on the function and structural features RsfS, as well as compared RsfS with MALSU1 in order to answer the emerging question on whether RsfS is a hibernation factor or a ribosome biogenesis factor. We believe that this review might promote future studies of the RsfS-involving molecular mechanisms, which so far remain completely unknown.

X-ray structure of the Rhodobacter sphaeroides reaction center with an M197 Phe→His substitution clarifies the properties of the mutant complex.

The first steps of the global process of photosynthesis take place in specialized membrane pigment-protein complexes called photosynthetic reaction centers (RCs). The RC of the photosynthetic purple bacterium Rhodobacter sphaeroides, a relatively simple analog of the more complexly organized photosystem II in plants, algae and cyanobacteria, serves as a convenient model for studying pigment-protein interactions that affect photochemical processes. In bacterial RCs the bacteriochlorophyll (BChl) dimer P serves as the primary electron donor, and its redox potential is a critical factor in the efficient functioning of the RC. It has previously been shown that the replacement of Phe M197 by His strongly affects the oxidation potential of P (E m P/P+), increasing its value by 125 mV, as well as increasing the thermal stability of RC and its stability in response to external pressure. The crystal structures of F(M197)H RC at high resolution obtained using various techniques presented in this report clarify the optical and electrochemical properties of the primary electron donor and the increased resistance of the mutant complex to denaturation. The electron-density maps are consistent with the donation of a hydrogen bond from the imidazole group of His M197 to the C2-acetyl carbonyl group of BChl PB. The formation of this hydrogen bond leads to a considerable out-of-plane rotation of the acetyl carbonyl group and results in a 1.2 Šshift of the O atom of this group relative to the wild-type structure. Besides, the distance between BChl PA and PB in the area of pyrrole ring I was found to be increased by up to 0.17 Å. These structural changes are discussed in association with the spectral properties of BChl dimer P. The electron-density maps strongly suggest that the imidazole group of His M197 accepts another hydrogen bond from the nearest water molecule, which in turn appears to form two more hydrogen bonds to Asn M195 and Asp L155. As a result of the F(M197)H mutation, BChl PB finds itself connected to the extensive hydrogen-bonding network that pre-existed in wild-type RC. Dissimilarities in the two hydrogen-bonding networks near the M197 and L168 sites may account for the different changes of the E m P/P+ in F(M197)H and H(L168)F RCs. The involvement of His M197 in the hydrogen-bonding network also appears to be related to stabilization of the F(M197)H RC structure. Analysis of the experimental data presented here and of the data available in the literature points to the fact that the hydrogen-bonding networks in the vicinity of BChl dimer P may play an important role in fine-tuning the redox properties of the primary electron donor.

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.

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.

Novel approaches for the lipid sponge phase crystallization of the Rhodobacter sphaeroides photosynthetic reaction center.

With the recent developments in the field of free-electron-laser-based serial femtosecond crystallography, the necessity to obtain a large number of high-quality crystals has emerged. In this work crystallization techniques were selected, tested and optimized for the lipid mesophase crystallization of the Rhodobacter sphaeroides membrane pigment-protein complex, known as the photosynthetic reaction center (RC). Novel approaches for lipid sponge phase crystallization in comparatively large volumes using Hamilton gas-tight glass syringes and plastic pipetting tips are described. An analysis of RC crystal structures obtained by lipid mesophase crystallization revealed non-native ligands that displaced the native electron-transfer cofactors (carotenoid sphero-idene and a ubi-quinone molecule) from their binding pockets. These ligands were identified and were found to be lipids that are major mesophase components. The selection of distinct co-crystallization conditions with the missing cofactors facilitated the restoration of sphero-idene in its binding site.

Cryo-EM structure of the ribosome functional complex of the human pathogen Staphylococcus aureus at 3.2 Å resolution.

Staphylococcus aureus is a bacterial pathogen and one of the leading causes of healthcare-acquired infections in the world. The growing antibiotic resistance of S. aureus obliges us to search for new drugs and treatments. As the majority of antibiotics target the ribosome, knowledge of its detailed structure is crucial for drug development. Here, we report the cryo-EM reconstruction at 3.2 Å resolution of the S. aureus ribosome with P-site tRNA, messenger RNA, and 10 RNA modification sites previously not assigned or visualized. The resulting model is the most precise and complete high-resolution structure to date of the S. aureus 70S ribosome with functional ligands.

Structure of the ribosomal P stalk base in archaean Methanococcus jannaschii.

Complexes of archaeal ribosomal proteins uL11 and uL10/P0 (the two-domain N-terminal fragment of uL10, uL10NTF/P0NTF) with the adjacent 74 nucleotides of 23S rRNA fragment (23SrRNA(74)) from Methanococcus jannaschii (Mja) were obtained, crystallized and their structures were studied. The comparative structural analysis of the complexes of Mja uL10NTF•23SrRNA(74) and Mja uL10NTF•uL11•23SrRNA(74) shows that the insertion of uL11 in the binary complex does not change the conformation of the 23S rRNA fragment. On the other hand, the interaction with this specific RNA fragment leads to the restructuring of uL11 compared to the structure of this protein in the free state. Besides, although analysis confirmed the mobility of uL10/P0 domain II, disproved the assumption that it may be in contact with rRNA or uL11. In addition, the Mja uL10NTF•uL11•23SrRNA(74) complex was cocrystallized with the antibiotic thiostrepton, and the structure of this complex was solved. The thiostrepton binding site in this archaeal complex was found between the 23S rRNA and the N-terminal domain (NTD) of the Mja uL11 protein, similar to its binding site in the one of bacterial ribosome complex with thiostrepton. Upon binding of thiostrepton, the NTD of uL11 shifts toward rRNA by 7 Å. Such a shift may be the cause of the inhibitory effect of the antibiotic on the recruitment of translation factors to the GTPase-activating region in archaeal ribosomes, similar to its inhibitory effect on protein synthesis in bacterial ribosomes.

The key role of E418 carboxyl group in the formation of Nt.BspD6I nickase active site: Structural and functional properties of Nt.BspD6I E418A mutant.

The mutated nickase Nt.BspD6I E418A has been obtained by site-directed mutagenesis. The purified protein has been crystallized, and its spatial structure has been determined at 2.45 Å resolution. An analysis of the crystal structures of the wild-type and mutated nickase have shown that the elimination of a carboxyl group due to the E418A mutation initiates marked conformational changes in both the N-terminal recognition domain and the C-terminal catalytic domain of nickase and insignificantly affects its linker domain. This is supported by changes in the functional properties of mutated nickase: an increase in the oligomerization capacity in the presence of a substrate, a reduction in the capacity to bind a substrate, and complete loss of catalytic activity.

Mechanism of ribosome shutdown by RsfS in Staphylococcus aureus revealed by integrative structural biology approach.

For the sake of energy preservation, bacteria, upon transition to stationary phase, tone down their protein synthesis. This process is favored by the reversible binding of small stress-induced proteins to the ribosome to prevent unnecessary translation. One example is the conserved bacterial ribosome silencing factor (RsfS) that binds to uL14 protein onto the large ribosomal subunit and prevents its association with the small subunit. Here we describe the binding mode of Staphylococcus aureus RsfS to the large ribosomal subunit and present a 3.2 Å resolution cryo-EM reconstruction of the 50S-RsfS complex together with the crystal structure of uL14-RsfS complex solved at 2.3 Å resolution. The understanding of the detailed landscape of RsfS-uL14 interactions within the ribosome shed light on the mechanism of ribosome shutdown in the human pathogen S. aureus and might deliver a novel target for pharmacological drug development and treatment of bacterial infections.