Development of a universal nanobody-binding Fab module for fiducial-assisted cryo-EM studies of membrane proteins.

With conformation-specific nanobodies being used for a wide range of structural, biochemical, and cell biological applications, there is a demand for antigen-binding fragments (Fabs) that specifically and tightly bind these nanobodies without disturbing the nanobody-target protein interaction. Here, we describe the development of a synthetic Fab (termed NabFab) that binds the scaffold of an alpaca-derived nanobody with picomolar affinity. We demonstrate that upon complementary-determining region grafting onto this parent nanobody scaffold, nanobodies recognizing diverse target proteins and derived from llama or camel can cross-react with NabFab without loss of affinity. Using NabFab as a fiducial and size enhancer (50 kDa), we determined the high-resolution cryogenic electron microscopy (cryo-EM) structures of nanobody-bound VcNorM and ScaDMT, both small membrane proteins of ∼50 kDa. Using an additional anti-Fab nanobody further facilitated reliable initial three-dimensional structure determination from small cryo-EM test datasets. Given that NabFab is of synthetic origin, is humanized, and can be conveniently expressed in Escherichia coli in large amounts, it may be useful not only for structural biology but also for biomedical applications.

An acetylatable lysine controls CRP function in E. coli.

Transcriptional regulation is the key to ensuring that proteins are expressed at the proper time and the proper amount. In Escherichia coli, the transcription factor cAMP receptor protein (CRP) is responsible for much of this regulation. Questions remain, however, regarding the regulation of CRP activity itself. Here, we demonstrate that a lysine (K100) on the surface of CRP has a dual function: to promote CRP activity at Class II promoters, and to ensure proper CRP steady state levels. Both functions require the lysine's positive charge; intriguingly, the positive charge of K100 can be neutralized by acetylation using the central metabolite acetyl phosphate as the acetyl donor. We propose that CRP K100 acetylation could be a mechanism by which the cell downwardly tunes CRP-dependent Class II promoter activity, whilst elevating CRP steady state levels, thus indirectly increasing Class I promoter activity. This mechanism would operate under conditions that favor acetate fermentation, such as during growth on glucose as the sole carbon source or when carbon flux exceeds the capacity of the central metabolic pathways.

Efficacy and safety of once-weekly dulaglutide versus insulin glargine in mainly Asian patients with type 2 diabetes mellitus on metformin and/or a sulphonylurea: A 52-week open-label, randomized phase III trial.

AIM: To compare the efficacy and safety of once-weekly dulaglutide with that of insulin glargine in combination with metformin and/or a sulphonylurea in mainly Asian patients with type 2 diabetes mellitus (T2DM). MATERIALS AND METHODS: In this 52-week, randomized, parallel-arm open-label study, we enrolled patients aged ≥18 years with T2DM for at least 6 months and a glycated haemoglobin (HbA1c) concentration ≥53.0 mmol/mol (7.0%) and ≤96.7 mmol/mol (11.0%). The primary outcome was change in HbA1c from baseline to week 26 to determine non-inferiority of dulaglutide 1.5 mg versus glargine. RESULTS: A total of 774 patients from China, South Korea, Mexico and Russia were randomly assigned (1:1:1) to dulaglutide 1.5 mg, dulaglutide 0.75 mg or glargine treatment groups. The patients' mean age was 55 years and the average T2DM duration was ~8 years. The least squares mean (SE) changes from baseline in HbA1c at 26 weeks were - 18.9 (0.73) mmol/mol (-1.73 [0.067]%) for dulaglutide 1.5 mg and -14.5 (0.73) mmol/mol (-1.33 [0.067]%) for dulaglutide 0.75 mg, compared with -12.7 (0.73) mmol/mol (-1.16 [0.067]%) for glargine. Statistical criteria for superiority were met with both dulaglutide 1.5 mg and dulaglutide 0.75 mg. More patients in the dulaglutide 1.5 and 0.75 mg groups achieved HbA1c target <53.0 mmol/mol (<7.0%) than in the glargine group at week 26 (P < 0.001 and P = 0.004, respectively). Body weight decreased with dulaglutide and increased with glargine. The incidence and rate of total hypoglycaemia were lower with dulaglutide versus glargine. Gastrointestinal adverse events, including diarrhoea and nausea, were the most frequently reported for patients taking dulaglutide. CONCLUSIONS: Once-weekly dulaglutide provides greater improvement in HbA1c, with weight loss and less hypoglycaemia, than once-daily insulin glargine in a population of mainly Asian patients with T2DM who had failed to achieve optimal glycaemic control on metformin and/or a sulphonylurea.

Structural characterization of the putative ABC-type 2 transporter from Thermotoga maritima MSB8.

This study describes the structure of the putative ABC-type 2 transporter TM0543 from Thermotoga maritima MSB8 determined at a resolution of 2.3 Å. In comparative sequence-clustering analysis, TM0543 displays similarity to NatAB-like proteins, which are components of the ABC-type Na(+) efflux pump permease. However, the overall structure fold of the predicted nucleotide-binding domain reveals that it is different from any known structure of ABC-type efflux transporters solved to date. The structure of the putative TM0543 domain also exhibits different dimer architecture and topology of its presumed ATP binding pocket, which may indicate that it does not bind nucleotide at all. Structural analysis of calcium ion binding sites found at the interface between TM0543 dimer subunits suggests that protein may be involved in ion-transporting activity. A detailed analysis of the protein sequence and structure is presented and discussed.

Structural characterization of a hypothetical protein: a potential agent involved in trimethylamine metabolism in Catenulispora acidiphila.

Catenulispora acidiphila is a newly identified lineage of actinomycetes that produces antimicrobial activities and represents a promising source of novel antibiotics and secondary metabolites. Among the discovered protein coding genes, 68 % were assigned a putative function, while the remaining 32 % are genes encoding "hypothetical" proteins. Caci_0382 is one of the "hypothetical" proteins that has very few homologs. Sequence analysis shows that the protein belongs to the NTF2-like protein family. The structure of Caci_0382 demonstrates that it shares the same fold and has a similar active site as limonene-1,2-epoxide hydrolase, which suggests that it may have a related function. Using a fluorescence thermal shift assay, we identified stabilizing compounds that suggest potential natural ligands of Caci_0382. Using this information, we determined the crystal structure in complex with trimethylamine to provide a better understanding of the function of this uncharacterized protein.

Large scale structural rearrangement of a serine hydrolase from Francisella tularensis facilitates catalysis.

Tularemia is a deadly, febrile disease caused by infection by the gram-negative bacterium, Francisella tularensis. Members of the ubiquitous serine hydrolase protein family are among current targets to treat diverse bacterial infections. Herein we present a structural and functional study of a novel bacterial carboxylesterase (FTT258) from F. tularensis, a homologue of human acyl protein thioesterase (hAPT1). The structure of FTT258 has been determined in multiple forms, and unexpectedly large conformational changes of a peripheral flexible loop occur in the presence of a mechanistic cyclobutanone ligand. The concomitant changes in this hydrophobic loop and the newly exposed hydrophobic substrate binding pocket suggest that the observed structural changes are essential to the biological function and catalytic activity of FTT258. Using diverse substrate libraries, site-directed mutagenesis, and liposome binding assays, we determined the importance of these structural changes to the catalytic activity and membrane binding activity of FTT258. Residues within the newly exposed hydrophobic binding pocket and within the peripheral flexible loop proved essential to the hydrolytic activity of FTT258, indicating that structural rearrangement is required for catalytic activity. Both FTT258 and hAPT1 also showed significant association with liposomes designed to mimic bacterial or human membranes, respectively, even though similar structural rearrangements for hAPT1 have not been reported. The necessity for acyl protein thioesterases to have maximal catalytic activity near the membrane surface suggests that these conformational changes in the protein may dually regulate catalytic activity and membrane association in bacterial and human homologues.

Antiviral Activity of Some Compounds of Gasteroid Mushrooms from Western Siberia.

The antiviral activity of aqueous and ethanol extracts from the fruiting bodies of gasteroid Basidiomy-cetes of Western Siberia: Lycoperdon pyriforme, Lycoperdon perlatum, and Phallus impudicus, as well as an aqueous extract from cultivated mycelium of P. impudicus and total polysaccharides from it, on MDCK cell culture against influenza A virus, was studied. Aqueous and ethanol extracts from the fruiting bodies of all studied gasteroid fungi showed antiviral activity against human influenza virus A/Aichi/2/68 (H3N2) and bird A/chicken/Kurgan/05/2005 virus (H5N1). At the same time, extracts from P. impudicus and L. pyriforme showed more pronouncing antiviral activity compared to the activity of the reference drug Tamiflu against the A/H5N1 avian influenza virus. A high antiviral efficacy of an aqueous extract from cultivated mycelium of the P. impudicus and a sample of total polysaccharides from this extract against the A/H5N1 avian influenza virus was revealed.

Substantial Antiviral Potential of Deoxyribozymes Fixed on Anatase Nanoparticles Against Influenza A Viruses in vitro and in vivo.

Influenza A viruses (IAV) are a high threat to humanity because of a lack of proper effective antiviral drugs and resistance of viruses to existing vaccines. We describe the sufficient anti-IAV effect of Ans/PL-Dz nanocomposites that contain deoxyribozymes (Dz) immobilized on anatase TiO2 nanoparticles (Ans) through polylysine linker (PL). The Dz-containing nanocomposites appear to be more efficient than the Ans/PL-ODN nanocomposites that contain common oligodeoxyribonucleotides (ODN) targeted to the same RNA regions of the viral genome. The simultaneous use of nanocomposites that contain Dz and ODN, which are targeted to different sites of viral RNA provides a higher overall effect than the independent action of each of them (synergism). The inhibition of IAV with the proposed nanocomposites was shown to be effective, sequence-specific, and dose-dependent. The most efficient Ans/PL-Dz nanocomposite exhibited a high antiviral effect in vivo on mice models. The efficiency of IAV inhibition with this nanocomposite in vitro and in vivo is higher than that for the approved antiflu drug oseltamivir. The results open the prospect of creating a unique antiviral agent suitable for IAV suppression.

Engineered antigen-binding fragments for enhanced crystallization of antibody:antigen complexes.

The atomic-resolution structural information that X-ray crystallography can provide on the binding interface between a Fab and its cognate antigen is highly valuable for understanding the mechanism of interaction. However, many Fab:antigen complexes are recalcitrant to crystallization, making the endeavor a considerable effort with no guarantee of success. Consequently, there have been significant steps taken to increase the likelihood of Fab:antigen complex crystallization by altering the Fab framework. In this investigation, we applied the surface entropy reduction strategy coupled with phage-display technology to identify a set of surface substitutions that improve the propensity of a human Fab framework to crystallize. In addition, we showed that combining these surface substitutions with previously reported Crystal Kappa and elbow substitutions results in an extraordinary improvement in Fab and Fab:antigen complex crystallizability, revealing a strong synergistic relationship between these sets of substitutions. Through comprehensive Fab and Fab:antigen complex crystallization screenings followed by structure determination and analysis, we defined the roles that each of these substitutions play in facilitating crystallization and how they complement each other in the process.

From TgO/GABA-AT, GABA, and T-263 Mutant to Conception of Toxoplasma.

Toxoplasma gondii causes morbidity, mortality, and disseminates widely via cat sexual stages. Here, we find T. gondii ornithine aminotransferase (OAT) is conserved across phyla. We solve TgO/GABA-AT structures with bound inactivators at 1.55 Å and identify an inactivator selective for TgO/GABA-AT over human OAT and GABA-AT. However, abrogating TgO/GABA-AT genetically does not diminish replication, virulence, cyst-formation, or eliminate cat's oocyst shedding. Increased sporozoite/merozoite TgO/GABA-AT expression led to our study of a mutagenized clone with oocyst formation blocked, arresting after forming male and female gametes, with "Rosetta stone"-like mutations in genes expressed in merozoites. Mutations are similar to those in organisms from plants to mammals, causing defects in conception and zygote formation, affecting merozoite capacitation, pH/ionicity/sodium-GABA concentrations, drawing attention to cyclic AMP/PKA, and genes enhancing energy or substrate formation in TgO/GABA-AT-related-pathways. These candidates potentially influence merozoite's capacity to make gametes that fuse to become zygotes, thereby contaminating environments and causing disease.

Acetylation of the response regulator RcsB controls transcription from a small RNA promoter.

Nε-lysine acetylation was recently discovered on many bacterial proteins that function in diverse cellular processes. Thus, many questions remain unanswered. For example, what mechanisms regulate lysine acetylation? Does acetylation affect physiology? To help answer these questions, we studied the Escherichia coli response regulator and transcription factor RcsB, which is reported to be acetylated in vitro. To characterize RcsB acetylation, we monitored transcription from the rprA promoter, which requires RcsB. The conventional view is that RcsB is activated by phosphorylation through either the Rcs phosphorelay or acetyl phosphate. We affirmed that rprA transcription requires phosphorylated RcsB and showed that acetyl-phosphate (AcP) is a phosphoryl group donor to RcsB. However, a mutant that accumulates AcP (ackA) exhibited a reduction in rprA transcription instead of the predicted increase. rprA transcription also diminished in the cobB mutant, which lacks the only known E. coli protein deacetylase. This suggests the existence of an inhibitory mechanism that involves lysine acetylation, a supposition supported by the observation that RcsB isolated from the ackA or cobB mutant was hyperacetylated. Finally, we used a genetic approach to identify an AckA- and CobB-sensitive lysine (Lys-154) that controls RcsB activity. We propose that acetylation inhibits RcsB activity and that some of this inhibition acts through the acetylation of Lys-154.

Impact of different Agrobacterium rhizogenes strains on secondary metabolites accumulation in Nitraria schoberi L. hairy roots and antiviral activity of their extracts against influenza virus of subtypes A (H5N1) and A (H3N2).

To optimize protocol for obtaining hairy roots of Nitraria schoberi L. with high antiviral activities, factors such as four strain types of Agrobacterium rhizogenes (A4, ATCC15834, R-1601, 8196), two explant types, namely cotyledonous and primary leaves of seedlings, and different cultivation durations (30 and 90 d) were studied. The formation of hairy roots was observed after 2 to 4 wk of incubation, depending on the type of explant and the strain of A. rhizogenes used. The maximum transformation frequency (85.7%) was observed in the cotyledons genetically modified with the strain ATCC15834. The transgenic nature of hairy roots was revealed by PCR with primers to the Agrobacterium oncogenes rolB and rolC. The absence of contamination of the culture by A. rhizogenes was confirmed by primers to the virC and virD1 genes. Phytochemical analysis showed that accumulation of individual metabolites in the line samples exceeded their levels in the native Nitraria roots. Catechin content in the cultures of long-term cultivation (90 d) was found 1.4 to 2.2 times higher than the same samples of short cultivation (30 d) and 4.8 to 10.8 times higher in comparison with the native roots. The most productive in terms of catechin level were hairy roots of long-term cultivation obtained during the transformation of primary leaves of N. schoberi seedlings with ATCC15834 strain. These data were consistent with the highest antiviral activities against influenza viruses of A (H5N1) and A (H3N2) subtypes with neutralization indexes 6.5 to 6.75 log10, and selectivity index values were in the range 15.4 to 16.4.

A high-throughput structural system biology approach to increase structure representation of proteins from Clostridioides difficile.

Clostridioides difficile causes life-threatening gastrointestinal infections. It is a high-risk pathogen due to a lack of effective treatments, antimicrobial resistance, and a poorly conserved genomic core. Herein, we report 30 X-ray structures from a structure genomics pipeline spanning 13 years, representing 10.2% of the X-ray structures for this important pathogen.

Crystal structures of putative phosphoglycerate kinases from B. anthracis and C. jejuni.

Phosphoglycerate kinase (PGK) is indispensable during glycolysis for anaerobic glucose degradation and energy generation. Here we present comprehensive structure analysis of two putative PGKs from Bacillus anthracis str. Sterne and Campylobacter jejuni in the context of their structural homologs. They are the first PGKs from pathogenic bacteria reported in the Protein Data Bank. The crystal structure of PGK from Bacillus anthracis str. Sterne (BaPGK) has been determined at 1.68 Å while the structure of PGK from Campylobacter jejuni (CjPGK) has been determined at 2.14 Å resolution. The proteins' monomers are composed of two domains, each containing a Rossmann fold, hinged together by a helix which can be used to adjust the relative position between two domains. It is also shown that apo-forms of both BaPGK and CjPGK adopt open conformations as compared to the substrate and ATP bound forms of PGK from other species.

Water Extract of the Chaga Medicinal Mushroom, Inonotus obliquus (Agaricomycetes), Inhibits SARS-CoV-2 Replication in Vero E6 and Vero Cell Culture Experiments.

The antiviral properties of water extracts from pharmaceutical raw materials of the chaga mushroom, Inonotus obliquus, were studied against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). All studies with infectious materials were carried out in an isolated virological laboratory of the State Research Center of Virology and Biotechnology Vector of Rospotrebnadzor, which has a sanitary and epidemiological conclusion for the right to work with pathogenic biological agents of I-II pathogenicity groups. Antiviral activity was determined by the ability of I. obliquus water extracts to inhibit the replication of SARS-CoV-2 (nCoV/Victoria /1/2020 strain) in Vero E6 and Vero cell cultures. The results of these studies showed that water extracts of I. obliquus are characterized by low toxicity in Vero and Vero E6 cell cultures and have antiviral activity against SARS-CoV-2. The 50% inhibitory concentration ranged from 0.75 to 11.6 µg/mL. A patent for the invention was received (Patent RU, 2741714 C 1, 2021).

Targeting a proteolytic neoepitope on CUB domain containing protein 1 (CDCP1) for RAS-driven cancers.

Extracellular proteolysis is frequently dysregulated in disease and can generate proteoforms with unique neoepitopes not found in healthy tissue. Here, we demonstrate that Abs that selectively recognize a proteolytic neoepitope on CUB domain containing protein 1 (CDCP1) could enable more effective and safer treatments for solid tumors. CDCP1 is highly overexpressed in RAS-driven cancers, and its ectodomain is cleaved by extracellular proteases. Biochemical, biophysical, and structural characterization revealed that the 2 cleaved fragments of CDCP1 remain tightly associated with minimal proteolysis-induced conformational change. Using differential phage display, we generated recombinant Abs that are exquisitely selective to cleaved CDCP1 with no detectable binding to the uncleaved form. These Abs potently targeted cleaved CDCP1-expressing cancer cells as an Ab-drug conjugate, an Ab-radionuclide conjugate, and a bispecific T cell engager. In a syngeneic pancreatic tumor model, these cleaved-specific Abs showed tumor-specific localization and antitumor activity with superior safety profiles compared with a pan-CDCP1 approach. Targeting proteolytic neoepitopes could provide an orthogonal "AND" gate for improving the therapeutic index.

Crystal structure of a putative transcriptional regulator SCO0520 from Streptomyces coelicolor A3(2) reveals an unusual dimer among TetR family proteins.

A structure of the apo-form of the putative transcriptional regulator SCO0520 from Streptomyces coelicolor A3(2) was determined at 1.8 Å resolution. SCO0520 belongs to the TetR family of regulators. In the crystal lattice, the asymmetric unit contains two monomers that form an Ω-shaped dimer. The distance between the two DNA-recognition domains is much longer than the corresponding distances in the known structures of other TetR family proteins. In addition, the subunits in the dimer have different conformational states, resulting in different relative positions of the DNA-binding and regulatory domains. Similar conformational modifications are observed in other TetR regulators and result from ligand binding. These studies provide information about the flexibility of SCO0520 molecule and its putative biological function.

Genome Features and In Vitro Activity against Influenza A and SARS-CoV-2 Viruses of Six Probiotic Strains.

PURPOSE: The aim of this work was to analyze the complete genome of probiotic bacteria Lactobacillus plantarum 8 RA 3, Lactobacillus fermentum 90 TC-4, Lactobacillus fermentum 39, Bifidobacterium bifidum 791, Bifidobacterium bifidum 1, and Bifidobacterium longum 379 and to test their activity against influenza A and SARS-CoV-2 viruses. METHODS: To confirm the taxonomic affiliation of the bacterial strains, MALDI TOF mass spectrometry and biochemical test systems were used. Whole genome sequencing was performed on the Illumina Inc. MiSeq platform. To determine the antiviral activity, A/Lipetsk/1V/2018 (H1N1 pdm09) (EPI_ISL_332798) and A/common gull/Saratov/1676/2018 (H5N6) (EPI_ISL_336925) influenza viruses and SARS-CoV-2 virus strain Australia/VIC01/2020 (GenBank: MT007544.1) were used. RESULTS: All studied probiotic bacteria are nonpathogenic for humans and do not contain the determinants of transmission-type antibiotic resistance and integrated plasmids. Resistance to antibiotics of different classes is explained by the presence of molecular efflux pumps of the MatE and MFS families. Cultures of L. fermentum 90 TC 4, L. plantarum 8 RA 3, and B. bifidum 791 showed a pronounced activity against influenza A viruses in MDCK cells. Activity against the SARS-CoV-2 virus was demonstrated only by the L. fermentum 90 TC 4 strain in VERO cells. CONCLUSIONS: The studied probiotic bacteria are safe, have antiviral activity, and are of great importance for the prevention of diseases caused by respiratory viruses that can also infect the human intestine.

In situ proteolysis for protein crystallization and structure determination.

We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.

In vitro temperature sensing with up-conversion NaYF4:Yb3+/Tm3+-based nanocomposites: Peculiarities and pitfalls.

The luminescence intensity ratio method, exploiting the temperature-dependent luminescence of the thermally coupled energy levels, is regarded as a very promising approach for optical temperature measurement at the cellular level. In this study, it was found that bare NaYF4:Yb3+/Tm3+ nanoparticles cannot be used as a cellular thermosensor in principle because of their tendency to aggregate, which significantly affects the luminescent properties of the complex, introducing uncertainty in the intensity ratio measurement. NaYF4:Yb3+/Tm3+ up-conversion nanoparticles, coated with polyethylene glycol (PEG) and carboxyl groups (COOH), on the other hand, proved to be promising candidates for the role of thermosensors. For the first time the temperature sensitivity of the NaYF4:Yb3+/Tm3+@PEG@COOH thermosensor was calculated in water and in biotissues. It was found that the sensitivity of the thermosensor increased by 1.3 times during the transition from water to egg white and urine - from 1.17% × K-1 to 1.58% × K-1. This effect is associated with the chemical composition of the studied media. The results obtained suggest that using upconversion nanocomplexes as primary thermosensors is still difficult.