AnnoDUF: A Web-Based Tool for Annotating Functions of Proteins Having Domains of Unknown Function.

The rapid expansion of biological sequence databases due to high-throughput genomic and proteomic sequencing methods has left a considerable number of identified protein sequences with unclear or incomplete functional annotations. Domains of unknown function (DUFs) are protein domains that lack functional annotations but are present in numerous proteins. To address the challenge of finding functional annotations for DUFs, we have developed a computational method that efficiently identifies and annotates these enigmatic protein domains by utilizing the position-specific iterative basic local alignment search tool (PSI-BLAST) and data mining techniques. Our pipeline identifies putative potential functionalities of DUFs, thereby decreasing the gap between known sequences and functions. The tool can also take user input sequences to annotate. We executed our pipeline on 5111 unique DUF sequences obtained from Pfam, resulting in putative annotations for 2007 of these. These annotations were subsequently incorporated into a comprehensive database and interfaced with a web-based server named "AnnoDUF". AnnoDUF is freely accessible to both academic and industrial users, via the World Wide Web at the link http://bts.ibab.ac.in/annoduf.php. All scripts used in this study are uploaded to the GitHub repository, and these can be accessed from https://github.com/BioToolSuite/AnnoDUF.

NAD+ pool depletion as a signal for the Rex regulon involved in Streptococcus agalactiae virulence.

In many Gram-positive bacteria, the redox-sensing transcriptional repressor Rex controls central carbon and energy metabolism by sensing the intra cellular balance between the reduced and oxidized forms of nicotinamide adenine dinucleotide; the NADH/NAD+ ratio. Here, we report high-resolution crystal structures and characterization of a Rex ortholog (Gbs1167) in the opportunistic pathogen, Streptococcus agalactiae, also known as group B streptococcus (GBS). We present structures of Rex bound to NAD+ and to a DNA operator which are the first structures of a Rex-family member from a pathogenic bacterium. The structures reveal the molecular basis of DNA binding and the conformation alterations between the free NAD+ complex and DNA-bound form of Rex. Transcriptomic analysis revealed that GBS Rex controls not only central metabolism, but also expression of the monocistronic rex gene as well as virulence gene expression. Rex enhances GBS virulence after disseminated infection in mice. Mechanistically, NAD+ stabilizes Rex as a repressor in the absence of NADH. However, GBS Rex is unique compared to Rex regulators previously characterized because of its sensing mechanism: we show that it primarily responds to NAD+ levels (or growth rate) rather than to the NADH/NAD+ ratio. These results indicate that Rex plays a key role in GBS pathogenicity by modulating virulence factor gene expression and carbon metabolism to harvest nutrients from the host.

Structures of two ArsR As(III)-responsive transcriptional repressors: Implications for the mechanism of derepression.

ArsR As(III)-responsive transcriptional repressors, members of the ArsR/SmtB family of metalloregulatory proteins, have been characterized biochemically but, to date, no As(III)-bound structure has been solved. Here we report two crystal structures of ArsR repressors from Acidithiobacillus ferrooxidans (AfArsR) and Corynebacterium glutamicum (CgArsR) in the As(III)-bound form. AfArsR crystallized in P21 space group and diffracted up to 1.86 Å. CgArsR crystallized in P212121 and diffracted up to 1.6 Å. AfArsR showed one As(III) bound in one subunit of the homodimer, while the CgArsR structure showed two As(III) bound with S3 coordination, one in each monomer. Previous studies indicated that in AfArsR As(III) binds to Cys95, Cys96 and Cys102 from the same monomer, while, in CgArsR, to Cys15, Cys16 from one monomer and Cys55 from the other monomer. The dimer interfaces of these structures showed distinct differences from other members of the ArsR/SmtB family of proteins, which potentially renders multiple options for evolving metal(loid) binding sites in this family of proteins. Also, CgArsR presents a new α2-N binding site, not the previously predicted α3-N site. Despite differences in the location of the binding cysteines in the primary sequences of these proteins, the two metal binding sites are almost congruent on their structures, an example of convergent evolution. Analyses of the electrostatic surface of the proteins at the DNA binding domain indicate that there two different modes of derepression in the ArsR/SmtB family of metalloregulatory proteins.

Regulatory Activities of Four ArsR Proteins in Agrobacterium tumefaciens 5A.

UNLABELLED: ArsR is a well-studied transcriptional repressor that regulates microbe-arsenic interactions. Most microorganisms have an arsR gene, but in cases where multiple copies exist, the respective roles or potential functional overlap have not been explored. We examined the repressors encoded by arsR1 and arsR2 (ars1 operon) and by arsR3 and arsR4 (ars2 operon) in Agrobacterium tumefaciens 5A. ArsR1 and ArsR4 are very similar in their primary sequences and diverge phylogenetically from ArsR2 and ArsR3, which are also quite similar to one another. Reporter constructs (lacZ) for arsR1, arsR2, and arsR4 were all inducible by As(III), but expression of arsR3 (monitored by reverse transcriptase PCR) was not influenced by As(III) and appeared to be linked transcriptionally to an upstream lysR-type gene. Experiments using a combination of deletion mutations and additional reporter assays illustrated that the encoded repressors (i) are not all autoregulatory as is typically known for ArsR proteins, (ii) exhibit variable control of each other's encoding genes, and (iii) exert variable control of other genes previously shown to be under the control of ArsR1. Furthermore, ArsR2, ArsR3, and ArsR4 appear to have an activator-like function for some genes otherwise repressed by ArsR1, which deviates from the well-studied repressor role of ArsR proteins. The differential regulatory activities suggest a complex regulatory network not previously observed in ArsR studies. The results indicate that fine-scale ArsR sequence deviations of the reiterated regulatory proteins apparently translate to different regulatory roles. IMPORTANCE: Given the significance of the ArsR repressor in regulating various aspects of microbe-arsenic interactions, it is important to assess potential regulatory overlap and/or interference when a microorganism carries multiple copies of arsR This study explores this issue and shows that the four arsR genes in A. tumefaciens 5A, associated with two separate ars operons, encode proteins exhibiting various degrees of functional overlap with respect to autoregulation and cross-regulation, as well as control of other functional genes. In some cases, differences in regulatory activity are associated with only limited differences in protein primary structure. The experiments summarized herein also present evidence that ArsR proteins appear to have activator functions, representing novel regulatory activities for ArsR, previously known only to be a repressor.

Identification and characterization of domain-specific inhibitors of DENV NS3 and NS5 proteins by in silico screening methods.

The dengue virus (DENV) infects approximately 400 million people annually worldwide causing significant morbidity and mortality. Despite advances in understanding the virus life cycle and infectivity, no specific treatment for this disease exists due to the lack of therapeutic drugs. In addition, vaccines available currently are ineffective with severe side effects. Therefore, there is an urgent need for developing therapeutics suitable for effective management of DENV infection. In this study, we adopted a drug repurposing strategy to identify new therapeutic use of existing FDA approved drug molecules to target DENV2 non-structural proteins NS3 and NS5 using computational approaches. We used Drugbank database molecules for virtual screening and multiple docking analysis against a total of four domains, the NS3 protease and helicase domains and NS5 MTase and RdRp domains. Subsequently, MD simulations and MM-PBSA analysis were performed to validate the intrinsic atomic interactions and the binding affinities. Furthermore, the internal dynamics in all four protein domains, in presence of drug molecule binding were assessed using essential dynamics and free energy landscape analyses, which were further coupled with conformational dynamics-based clustering studies and cross-correlation analysis to map the regions that exhibit these structural variations. Our comprehensive analysis identified tolcapone, cefprozil, delavirdine and indinavir as potential inhibitors of NS5 MTase, NS5 RdRp, NS3 protease and NS3 helicase functions, respectively. These high-confidence candidate molecules will be useful for developing effective anti-DENV therapy to combat dengue infection.Communicated by Ramaswamy H. Sarma.

Diagnostic and prophylactic potential of a stabilized foot-and-mouth disease serotype Asia1 virus like particles designed through a structure guided approach.

Intact capsids of foot-and-mouth disease virus (FMDV) play a vital role in eliciting a protective immune response. Any change in the physico-chemical environment of the capsids results in dissociation and poor immunogenicity. Structural bioinfomatics studies have been carried out to predict the amino acids at the interpentameric region that resulted in the identification of mutant virus-like particles(VLPs) of FMDV serotype Asia1/IND/63/1972. The insect cell expressed VLPs were evaluated for their stability by sandwich ELISA. Among 10 mutants, S93H showed maximum retention of antigenicity at different temperatures, indicating its higher thermal stability as revealed by the in-silico analysis and retained the antigenic sites of the virus demonstrated by Sandwich ELISA. The concordant results of the liquid phase blocking ELISA for estimation of antibody titre of known sera with stable mutant VLP as antigen in place of virus antigen demonstrate its diagnostic potential. The stable mutant VLP elicited a robust immune response with 85.6 % protection in guinea pigs against virus challenge. The stabilized VLP based antigen requires minimum biosafety and cold storage for production and transit besides, complying with differentiation of infected from vaccinated animals. It can effectively replace the conventional virus handling during antigen production for prophylactic and diagnostic use.

Dietary factors and SARS-CoV-2 contagion: in silico studies on modulation of viral and host proteins by spice actives.

The SARS-CoV-2 contagion has had a huge impact on world population. It has been observed that despite massive spread of the contagion in India particularly during the second wave, the overall case fatality rates remain low. This prompted us to look into dietary factors that can possibly modulate the viral impact and/or host response. In silico studies were carried out on forty-two commonly used spices and their 637 known active compounds with an aim of identifying such compounds that may have propensity to reduce viral impact or boost host immune response. We chose to study SARS-Cov-2 helicase on account of its functional importance in maintaining viral load within the host, and the human tank binding protein (TBK1) for its important role in host immunity. We carried out in silico virtual screening, docking studies with 637 phytochemical against these two proteins, using in silico methods. Upon assessing the strength of the ligand-target interactions and post simulation binding energy profile, our study identifies procyanidin-B4 from bay leaf, fenugreekine from fenugreek seed and gallotannin from pomegranate seed as active interactors that docked to viral helicase. Similarly, we identified eruboside B from garlic, gallotannin from pomegranate seed, as strong interacting partners to human TBK1. Our studies thus present dietary spice constituents as potential protagonists for further experimentation to understand how spices in the diet might help the hosts in countering the viral assault and mount a robust protective response against COVID and other infections.Communicated by Ramaswamy H. Sarma.

Traditional medicinal plants against replication, maturation and transmission targets of SARS-CoV-2: computational investigation.

COVID-19 is an infectious pandemic caused by the SARS-CoV-2 virus. The critical components of SARS-CoV-2 are the spike protein (S-protein) and the main protease (Mpro). Mpro is required for the maturation of the various polyproteins involved in replication and transcription. S-protein helps the SARS-CoV-2 to enter the host cells through the angiotensin-converting enzyme 2 (ACE2). Since ACE2 is required for the binding of SARS-CoV-2 on the host cells, ACE2 inhibitors and blockers have got wider attention, in addition to S-protein and Mpro modulators as potential therapeutics for COVID-19. So far, no specific drugs have shown promising therapeutic potential against COVID-19. The current study was undertaken to evaluate the therapeutic potential of traditional medicinal plants against COVID-19. The bioactives from the medicinal plants, along with standard drugs, were screened for their binding against S-protein, Mpro and ACE2 targets using molecular docking followed by molecular dynamics. Based on the higher binding affinity compared with standard drugs, bioactives were selected and further analyzed for their pharmacological properties such as drug-likeness, ADME/T-test, biological activities using in silico tools. The binding energies of several bioactives analyzed with target proteins were relatively comparable and even better than the standard drugs. Based on Lipinski factors and lower binding energies, seven bioactives were further analyzed for their pharmacological and biological characteristics. The selected bioactives were found to have lower toxicity with a higher GI absorption rate and potent anti-inflammatory and anti-viral activities against targets of COVID-19. Therefore, the bioactives from these medicinal plants can be further developed as phytopharmaceuticals for the effective treatment of COVID-19.Communicated by Ramaswamy H. Sarma.

Purification, crystallization and preliminary X-ray diffraction studies of the arsenic repressor ArsR from Corynebacterium glutamicum.

ArsR is a member of the SmtB/ArsR family of metalloregulatory proteins that regulate prokaryotic arsenic-resistance operons. Here, the crystallization and preliminary X-ray diffraction studies of a cysteine-free derivative of ArsR from Corynebacterium glutamicum (CgArsR-C15/16/55S) are reported. CgArsR-C15/16/55S was expressed, purified, crystallized and X-ray diffraction data were collected to 1.86 Å resolution. The protein crystallized in a tetragonal space group (P4), with unit-cell parameters a = b = 41.84, c = 99.47 Å.

Identification of a repurposed drug as an inhibitor of Spike protein of human coronavirus SARS-CoV-2 by computational methods.

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) is an emerging new viral pathogen that causes severe respiratory disease. SARS-CoV-2 is responsible for the outbreak of COVID-19 pandemic worldwide. As there are no confirmed antiviral drugs or vaccines currently available for the treatment of COVID-19, discovering potent inhibitors or vaccines are urgently required for the benefit of humanity. The glycosylated Spike protein (S-protein) directly interacts with human angiotensin-converting enzyme 2 (ACE2) receptor through the receptor-binding domain (RBD) of S-protein. As the S-protein is exposed to the surface and is essential for entry into the host, the S-protein can be considered as a first-line therapeutic target for antiviral therapy and vaccine development. In silico screening, docking, and molecular dynamics simulation studies were performed to identify repurposing drugs using DrugBank and PubChem library against the RBD of S-protein. The study identified a laxative drug, Bisoxatin (DB09219), which is used for the treatment of constipation and preparation of the colon for surgical procedures. It binds nicely at the S-protein-ACE2 interface by making substantial π-π interactions with Tyr505 in the 'Site 1' hook region of RBD and hydrophilic interactions with Glu406, Ser494, and Thr500. Bisoxatin consistently binds to the protein throughout the 100 ns simulation. Taken together, we propose that the discovered molecule, Bisoxatin may be a promising repurposable drug molecule to develop new chemical libraries for inhibiting SARS-CoV-2 entry into the host.

Genomewide characterisation of the genetic diversity of carotenogenesis in bacteria of the order Sphingomonadales.

The order Sphingomonadales is a taxon of bacteria with a variety of physiological features and carotenoid pigments. Some of the coloured strains within this order are known to be aerobic anoxygenic phototrophs that contain characteristic photosynthesis gene clusters (PGCs). Previous work has shown that majority of the ORFs putatively involved in the biosynthesis of C40 carotenoids are located outside the PGCs in these strains. The main purpose of this study was to understand the genetic basis for the various colour/carotenoid phenotypes of the strains of Sphingomonadales. Comparative analyses of the genomes of 41 strains of this order revealed that there were different patterns of clustering of carotenoid biosynthesis (crt) ORFs, with four ORF clusters being the most common. The analyses also revealed that co-occurrence of crtY and crtI is an evolutionarily conserved feature in Sphingomonadales and other carotenogenic bacteria. The comparisons facilitated the categorisation of bacteria of this order into four groups based on the presence of different crt ORFs. Yellow coloured strains most likely accumulate nostoxanthin, and contain six ORFs (group I: crtE, crtB, crtI, crtY, crtZ, crtG). Orange coloured strains may produce adonixanthin, astaxanthin, canthaxanthin and erythroxanthin, and contain seven ORFs (group II: crtE, crtB, crtI, crtY, crtZ, crtG, crtW). Red coloured strains may accumulate astaxanthin, and contain six ORFs (group III: crtE, crtB, crtI, crtY, crtZ, crtW). Non-pigmented strains may contain a smaller subset of crt ORFs, and thus fail to produce any carotenoids (group IV). The functions of many of these ORFs remain to be characterised.

Identification of New Inhibitors for Human SIRT1: An in-silico Approach.

BACKGROUND: Human SIRT1 is a class III histone deacetylase (HDAC) family protein. As the overexpression of hSIRT1 leads to cancer, inhibiting its HDAC function may be a better strategy for the treatment of cancer. Till now, only a few reported inhibitor compounds have reached the stage of animal studies; hence, identifying high efficacy inhibitors of hSIRT1 is essential. OBJECTIVE: The main objective of the study is to obtain a new class of inhibitor compounds of hSIRT1 by the rational structure-based method. METHODOLOGY: We performed virtual screening using AutoDock Vina for the HDAC domain of hSIRT1 against the Drug- Bank library containing 1,716 compounds. The recently determined crystal structure of the HDAC domain of hSIRT1 (PDB Id: 4KXQ) was used for docking studies. Subsequently, we performed molecular dynamics simulations and an invitro deacetylase assay for selected compounds. RESULTS: Virtual screening studies yielded seven compounds from two chemical classes, namely diphenyl and oxycoumarin derivatives. Molecular dynamic simulations confirmed that the predicted seven compounds bind well to their respective complex structures. Moreover, four commercially available drugs containing the predicted compounds showed significant inhibition of hSIRT1 deacetylase activity in comparison to the known hSIRT1 inhibitor (sirtinol). CONCLUSION: Our results indicate that the compounds of the diphenyl and oxycoumarin series may serve as useful scaffolds in the development of new chemical libraries of hSIRT1 inhibitory activity.

Mutations in ARSB in MPS VI patients in India.

Mucopolysaccharidosis VI (MPS VI) is an autosomal recessive inborn error of metabolism caused by mutations in the arylsulfatase B gene (ARSB) and consequent deficient activity of ARSB, a lysosomal enzyme. We present here the results of a study undertaken to identify the mutations in ARSB in MPS VI patients in India. Around 160 ARSB mutations, of which just 4 are from India, have been reported in the literature. Our study covered nine MPS VI patients from eight families. Both familial mutations were found in seven families, and only one mutation was found in one family. Seven mutations were found - four novel (p.G38_G40del3, p.C91R, p.L98R and p.R315P), two previously reported from India (p.D53N and p.W450C), and one reported from outside India (p.R160Q). One mutation, p.W450C, was present in two families, and the other six mutations were present in one family each. Analysis of the molecular structure of the enzyme revealed that most of these mutations either cause loss of an active site residue or destabilize the structure of the enzyme. The only previous study on mutations in ARSB in Indian MPS VI patients, by Kantaputra et al. 2014 [1], reported four novel mutations of which two (p.D53N and p.W450C) were found in our study as well. Till date, nine mutations have been reported from India, through our study and the Kantaputra study. Eight out of these nine mutations have been found only in India. This suggests that the population studied by us might have its own typical set of mutations, with other populations equally likely to have their own set of mutations.

Role of bound Zn(II) in the CadC Cd(II)/Pb(II)/Zn(II)-responsive repressor.

The Staphylococcus aureus plasmid pI258 cadCA operon encodes a P-type ATPase, CadA, that confers resistance to Cd(II)/Pb(II)/Zn(II). Expression is regulated by CadC, a homodimeric repressor that dissociates from the cad operator/promoter upon binding of Cd(II), Pb(II), or Zn(II). CadC is a member of the ArsR/SmtB family of metalloregulatory proteins. The crystal structure of CadC shows two types of metal binding sites, termed Site 1 and Site 2, and the homodimer has two of each. Site 1 is the physiological inducer binding site. The two Site 2 metal binding sites are formed at the dimerization interface. Site 2 is not regulatory in CadC but is regulatory in the homologue SmtB. Here the role of each site was investigated by mutagenesis. Both sites bind either Cd(II) or Zn(II). However, Site 1 has higher affinity for Cd(II) over Zn(II), and Site 2 prefers Zn(II) over Cd(II). Site 2 is not required for either derepression or dimerization. The crystal structure of the wild type with bound Zn(II) and of a mutant lacking Site 2 was compared with the SmtB structure with and without bound Zn(II). We propose that an arginine residue allows for Zn(II) regulation in SmtB and, conversely, a glycine results in a lack of regulation by Zn(II) in CadC. We propose that a glycine residue was ancestral whether the repressor binds Zn(II) at a Site 2 like CadC or has no Site 2 like the paralogous ArsR and implies that acquisition of regulatory ability in SmtB was a more recent evolutionary event.

Aquaglyceroporins: ancient channels for metalloids.

The identification of aquaglyceroporins as uptake channels for arsenic and antimony shows how these toxic elements can enter the food chain, and suggests that food plants could be genetically modified to exclude arsenic while still accumulating boron and silicon.

Evolution of metal(loid) binding sites in transcriptional regulators.

Expression of the genes for resistance to heavy metals and metalloids is transcriptionally regulated by the toxic ions themselves. Members of the ArsR/SmtB family of small metalloregulatory proteins respond to transition metals, heavy metals, and metalloids, including As(III), Sb(III), Cd(II), Pb(II), Zn(II), Co(II), and Ni(II). These homodimeric repressors bind to DNA in the absence of inducing metal(loid) ion and dissociate from the DNA when inducer is bound. The regulatory sites are often three- or four-coordinate metal binding sites composed of cysteine thiolates. Surprisingly, in two different As(III)-responsive regulators, the metalloid binding sites were in different locations in the repressor, and the Cd(II) binding sites were in two different locations in two Cd(II)-responsive regulators. We hypothesize that ArsR/SmtB repressors have a common backbone structure, that of a winged helix DNA-binding protein, but have considerable plasticity in the location of inducer binding sites. Here we show that an As(III)-responsive member of the family, CgArsR1 from Corynebacterium glutamicum, binds As(III) to a cysteine triad composed of Cys(15), Cys(16), and Cys(55). This binding site is clearly unrelated to the binding sites of other characterized ArsR/SmtB family members. This is consistent with our hypothesis that metal(loid) binding sites in DNA binding proteins evolve convergently in response to persistent environmental pressures.