Integrated approaches for the recognition of small molecule inhibitors for Toll-like receptor 4.

Toll-like receptors (TLRs) are pattern recognition receptors present on the surface of cells playing a crucial role in innate immunity. One of the TLRs, TLR4, recognizes LPS (Lipopolysaccharide) as its ligand leading to the release of anti-inflammatory mediators as well as pro-inflammatory cytokines through signal transduction and domain recruitment. TLR4 homodimerizes at its intracellular TIR (Toll/interleukin-1 receptor) domain that helps in the recruitment of the TRAM/TICAM2 (TIR domain-containing adaptor molecule 2) molecule. TRAM also contains TIR domain which in turn, dimerizes and functions as an adapter protein to further recruit TRIF/TICAM1 (TIR domain-containing adaptor molecule 1) protein for mediating downstream signaling. Apart from LPS, TLR4 also recognizes endogenous ligands like fibrinogen, HMGB1, and hyaluronan in autoimmune conditions and sepsis. We employed computational approaches to target TRAM and recognize small molecule inhibitors from small molecules of natural origin, as contained in the Super Natural II database. Finally, cell reporter assays and NMR studies enabled the identification of promising lead compounds. Hence, this study aims to attenuate the signaling of the TLR4-TRAM-TRIF cascade in these auto-inflammatory conditions.

Computational analysis of the flexibility in the disordered linker region connecting LIM domains in cysteine-glycine-rich protein.

One of the key proteins that are present in the Z-disc of cardiac tissues, CSRP3, has been implicated in dilated and hypertrophic cardiomyopathy leading to heart failure. Although multiple cardiomyopathy-related mutations have been reported to reside on the two LIM domains and the disordered regions connecting the domains in this protein, the exact role of the disordered linker region is not clear. The linker harbors a few post-translational modification sites and is expected to be a regulatory site. We have carried out evolutionary studies on 5614 homologs spanning across taxa. We also performed molecular dynamics simulations of full-length CSRP3 to show that the length variations and conformational flexibility of the disordered linker could provide additional levels of functional modulation. Finally, we show that the CSRP3 homologs with widely different lengths of the linker regions could display diversity in their functional specifications. The present study provides a useful perspective to our understanding of the evolution of the disordered region between CSRP3 LIM domains.

Structure modelling of odorant receptor from Aedes aegypti and identification of potential repellent molecules.

Odorant receptors (ORs) are important class of proteins involved in olfactory behaviour of insects. These are GPCR-like heptahelical transmembrane proteins with inverted topology compared to GPCR and require a co-receptor (ORco) for their function. OR function can be modulated through small molecules and negative modulation can be beneficial in case of disease vectors like Aedes aegypti. OR4 of A. aegypti is implicated in host recognition through human odour. Aedes aegypti is a vector for viruses that spread diseases like dengue, Zika and Chikungunya. In this study, we have attempted to model the full-length structure of OR4 and the ORco of A. aegypti due to lack of experimental structure. Further, we have screened a library of natural compounds (>0.3 million) along with known repellent molecules against ORco and OR4. Many natural compounds, including those from plants like Ocimum tenuiflorum (Holy Basil) and Piper nigrum (Black pepper), were found to have better binding affinity towards ORco compared to known repellents like DEET providing an alternative to existing repellent molecules. For specific inhibitor of OR4, several natural compounds (including those from plant like Mulberry) were identified. Further, we have utilized multiple docking approaches and conservation analysis to understand the interaction between OR4 and ORco. It was observed that the residues from the seventh transmembrane helix of OR4 and pore forming helix of ORco could play an important role along with known intracellular loop 3 residues in mediating the heteromer formation of OR and ORco.

Structural modelling and dynamics of full-length of TLR10 sheds light on possible modes of dimerization, ligand binding and mechanism of action.

Toll like receptors (TLRs) play a pivotal role in innate and adaptive immunity. There are 10 TLRs in the human genome, of which TLR10 is the least characterized. Genetic polymorphism of TLR10 has been shown to be associated with multiple diseases including tuberculosis and rheumatoid arthritis. TLR10 consists of an extracellular domain (ECD), a single-pass transmembrane (TM) helix and intracellular TIR (Toll/Interleukin-1 receptor) domain. ECD is employed for ligand recognition and the intracellular domain interacts with other TIR domain-containing adapter proteins for signal transduction. Experimental structure of ECD or TM domain is not available for TLR10. In this study, we have modelled multiple forms of TLR10-ECD dimers, such as closed and open forms, starting from available structures of homologues. Subsequently, multiple full-length TLR10 homodimer models were generated by utilizing homology modelling and protein-protein docking. The dynamics of these models in membrane-aqueous environment revealed the global motion of ECD and TIR domain towards membrane bilayer. The TIR domain residues exhibited high root mean square fluctuation compared to ECD. The 'closed form' model was observed to be energetically more favorable than 'open form' model. The evaluation of persistent interchain interactions, along with their conservation score, unveiled critical residues for each model. Further, the binding of dsRNA to TLR10 was modelled by defined and blind docking approaches. Differential binding of dsRNA to the protomers of TLR10 was observed upon simulation that could provide clues on ligand disassociation. Dynamic network analysis revealed that the 'open form' model can be the functional form while 'closed form' model can be the apo form of TLR10.

Biography of a scientist with strength, substance, sincerity and service: Late N. Srinivasan (1962-2021).

Late N. Srinivasan belongs to the GN Ramachandran lineage of protein structural analysts. His role in the advancement of the structure based understanding of signal transduction, protein kinase analyses and host-pathogen interactions both developing and using Bioinformatics tools for protein-protein interactions, protein dynamics, remote homology detection and polypeptide stereochemistry is well documented in the literature. Thus, his contribution to the understanding of protein function through structural analysis, using computational models and tools, is exceptional.

Chronic exposure of humans to high level natural background radiation leads to robust expression of protective stress response proteins.

Understanding exposures to low doses of ionizing radiation are relevant since most environmental, diagnostic radiology and occupational exposures lie in this region. However, the molecular mechanisms that drive cellular responses at these doses, and the subsequent health outcomes, remain unclear. A local monazite-rich high level natural radiation area (HLNRA) in the state of Kerala on the south-west coast of Indian subcontinent show radiation doses extending from ≤ 1 to ≥ 45 mGy/y and thus, serve as a model resource to understand low dose mechanisms directly on healthy humans. We performed quantitative discovery proteomics based on multiplexed isobaric tags (iTRAQ) coupled with LC-MS/MS on human peripheral blood mononuclear cells from HLNRA individuals. Several proteins involved in diverse biological processes such as DNA repair, RNA processing, chromatin modifications and cytoskeletal organization showed distinct expression in HLNRA individuals, suggestive of both recovery and adaptation to low dose radiation. In protein-protein interaction (PPI) networks, YWHAZ (14-3-3ζ) emerged as the top-most hub protein that may direct phosphorylation driven pro-survival cellular processes against radiation stress. PPI networks also identified an integral role for the cytoskeletal protein ACTB, signaling protein PRKACA; and the molecular chaperone HSPA8. The data will allow better integration of radiation biology and epidemiology for risk assessment [Data are available via ProteomeXchange with identifier PXD022380].

Quaternary variations in the structural assembly of N-acetylglucosamine-6-phosphate deacetylase from Pasteurella multocida.

N-acetylglucosamine 6-phosphate deacetylase (NagA) catalyzes the conversion of N-acetylglucosamine-6-phosphate to glucosamine-6-phosphate in amino sugar catabolism. This conversion is an essential step in the catabolism of sialic acid in several pathogenic bacteria, including Pasteurella multocida, and thus NagA is identified as a potential drug target. Here, we report the unique structural features of NagA from P. multocida (PmNagA) resolved to 1.95 Å. PmNagA displays an altered quaternary architecture with unique interface interactions compared to its close homolog, the Escherichia coli NagA (EcNagA). We confirmed that the altered quaternary structure is not a crystallographic artifact using single particle electron cryo-microscopy. Analysis of the determined crystal structure reveals a set of hot-spot residues involved in novel interactions at the dimer-dimer interface. PmNagA binds to one Zn2+ ion in the active site and demonstrates kinetic parameters comparable to other bacterial homologs. Kinetic studies reveal that at high substrate concentrations (~10-fold the KM ), the tetrameric PmNagA displays hysteresis similar to its distant neighbor, the dimeric Staphylococcus aureus NagA (SaNagA). Our findings provide key information on structural and functional properties of NagA in P. multocida that could be utilized to design novel antibacterials.

Homology Modeling Identifies Crucial Amino-Acid Residues That Confer Higher Na+ Transport Capacity of OcHKT1;5 from Oryza coarctata Roxb.

HKT1;5 loci/alleles are important determinants of crop salinity tolerance. HKT1;5s encode plasmalemma-localized Na+ transporters, which move xylem Na+ into xylem parenchyma cells, reducing shoot Na+ accumulation. Allelic variation in rice OsHKT1;5 sequence in specific landraces (Nona Bokra OsHKT1;5-NB/Nipponbare OsHKT1;5-Ni) correlates with variation in salt tolerance. Oryza coarctata, a halophytic wild rice, grows in fluctuating salinity at the seawater-estuarine interface in Indian and Bangladeshi coastal regions. The distinct transport characteristics of the shoots and roots expressing the O. coarctata OcHKT1;5 transporter are reported vis-à-vis OsHKT1;5-Ni. Yeast sodium extrusion-deficient cells expressing OcHKT1;5 are sensitive to increasing Na+ (10-100 mM). Electrophysiological measurements in Xenopus oocytes expressing O. coarctata or rice HKT1;5 transporters indicate that OcHKT1;5, like OsHKT1;5-Ni, is a Na+-selective transporter, but displays 16-fold lower affinity for Na+ and 3.5-fold higher maximal conductance than OsHKT1;5-Ni. For Na+ concentrations >10 mM, OcHKT1;5 conductance is higher than that of OsHKT1;5-Ni, indicating the potential of OcHKT1;5 for increasing domesticated rice salt tolerance. Homology modeling/simulation suggests that four key amino-acid changes in OcHKT1;5 (in loops on the extracellular side; E239K, G207R, G214R, L363V) account for its lower affinity and higher Na+ conductance vis-à-vis OsHKT1;5-Ni. Of these, E239K in OcHKT1;5 confers lower affinity for Na+ transport, as evidenced by Na+ transport assays of reciprocal site-directed mutants for both transporters (OcHKT1;5-K239E, OsHKT1;5-Ni-E270K) in Xenopus oocytes. Both transporters have likely analogous roles in xylem sap desalinization, and differences in xylem sap Na+ concentrations in both species are attributed to differences in Na+ transport affinity/conductance between the transporters.

EcRBPome: a comprehensive database of all known E. coli RNA-binding proteins.

The repertoire of RNA-binding proteins (RBPs) in bacteria play a crucial role in their survival, and interactions with the host machinery, but there is little information, record or characterisation in bacterial genomes. As a first step towards this, we have chosen the bacterial model system Escherichia coli, and organised all RBPs in this organism into a comprehensive database named EcRBPome. It contains RBPs recorded from 614 complete E. coli proteomes available in the RefSeq database (as of October 2018). The database provides various features related to the E. coli RBPs, like their domain architectures, PDB structures, GO and EC annotations etc. It provides the assembly, bioproject and biosample details of each strain, as well as cross-strain comparison of occurrences of various RNA-binding domains (RBDs). The percentage of RBPs, the abundance of the various RBDs harboured by each strain have been graphically represented in this database and available alongside other files for user download. To the best of our knowledge, this is the first database of its kind and we hope that it will be of great use to the biological community.

Correction: Population Distribution Analyses Reveal a Hierarchy of Molecular Players Underlying Parallel Endocytic Pathways.

[This corrects the article DOI: 10.1371/journal.pone.0100554.].

Evaluating Ylehd, a recombinant epoxide hydrolase from Yarrowia lipolytica as a potential biocatalyst for the resolution of benzyl glycidyl ether.

Glycidyl ethers and their vicinal diols are important building blocks in the organic synthesis of anti-cancer and anti-obesity drugs. Ylehd, an epoxide hydrolase from tropical marine yeast Yarrowia lipolytica, was explored for its enantioselective properties by kinetic, thermodynamic and in silico studies. Kinetic resolution of racemic phenyl glycidyl ether (PGE) yielded (S)-epoxide while for benzyl glycidyl ether (BGE) (R)-epoxide was obtained, with vicinal diols of the opposite configuration. Amongst the enantiomers of PGE and BGE, the (S)-selective conversion of benzyl glycidyl ether to its corresponding diol, (S)-3-benzyloxy-1,2-propanediol while retaining (R)-BGE was most favourable with 95% ee in 20 min. Enantioselective conversion of specific enantiomer of BGE to its corresponding diols was attributed to the favourable kinetic and thermodynamic parameters as well as to the number and proximity of water molecules near the base H325 in the active site pocket. The easily available and highly active Ylehd could be a potential biocatalyst for large scale preparation of pharmaceutically relevant chiral (R)-benzyl glycidyl ether and (S)-3-benzyloxy-1,2-propanediol.

Computational genome-wide survey of odorant receptors from two solitary bees Dufourea novaeangliae (Hymenoptera: Halictidae) and Habropoda laboriosa (Hymenoptera: Apidae).

Olfactory/odorant receptors (ORs) probably govern eusocial behaviour in honey bees through detection of cuticular hydrocarbons (CHCs) and queen mandibular gland pheromones (QMP). CHCs are involved in nest-mate recognition whereas QMP acts as sex pheromone for drones and as retinue pheromone for female workers. Further studies on the effect of eusociality on the evolution of ORs are hindered by the non-availability of comprehensive OR sets of solitary species. We report complete OR repertoires from two solitary bees Dufourea novaeangliae (112 ORs) and Habropoda laboriosa (151 ORs). We classify these ORs into 34 phylogenetic clades/subfamilies. Differences in the OR sets of solitary and eusocial bees are observed in individual subfamilies like subfamily 9-exon (putative CHC receptors) and L (contains putative QMP receptor group). A subfamily (H) including putative floral scent receptors is expanded in the generalist honey bees only, but not in the specialists. On the contrary, subfamily J is expanded in all bees irrespective of their degree of social complexity or food preferences. Finally, we show species-lineage specific and OR-subfamily specific differences in the putative cis-regulatory DNA motifs of the ORs from six hymenopteran species. Out of these, [A/G]CGCAAGCG[C/T] is a candidate master transcription factor binding site for multiple olfactory genes.

A tale of two paralogs: human Transformer2 proteins with differential RNA-binding affinities.

The Transformer2 (Tra2) proteins in humans are homologues of the Drosophila Tra2 protein. One of the two RNA-binding paralogs, Tra2ß, has been very well-studied over the past decade, but not much is known about Tra2α. It was very recently shown that the two proteins demonstrate the phenomenon of paralog compensation. Here, we provide a structural basis for this genetic backup circuit, using molecular modelling and dynamics studies. We show that the two proteins display similar binding specificities, but differential affinities to a short GAA-rich RNA stretch. Starting from the 6-nucleotide RNA in the solution structure, close to 4000 virtual mutations were modelled on RNA and the domain-RNA interactions were studied after energy minimisation to convergence. Separately, another known 13-nucleotide stretch was docked and the domain-RNA interactions were observed through a 100-ns dynamics trajectory. We have also demonstrated the 'compensatory' mechanism at the level of domains in one of the domain repeat-containing RNA-binding proteins.

Molecular modelling of human 5-hydroxytryptamine receptor (5-HT2A) and virtual screening studies towards the identification of agonist and antagonist molecules.

The serotonin receptors, also known as 5-hydroxytryptamine (5-HT) receptors, are a group of G protein-coupled receptors (GPCRs) and ligand-gated ion channels found in the central and peripheral nervous systems. GPCRs have a characteristic feature of activating different signalling pathways upon ligand binding and these ligands display several efficacy levels to differentially activate the receptor. GPCRs are primary drug targets due to their central role in several signal transduction pathways. Drug design for GPCRs is also most challenging due to their inherent promiscuity in ligand recognition, which gives rise to several side effects of existing drugs. Here, we have performed the ligand interaction study using the two prominent states of GPCR, namely the active and inactive state of the 5-HT2A receptor. Active state of 5-HT2A receptor model enhances the understanding of conformational difference which influences the ligand-binding site. A 5-HT2A receptor active state model was constructed by homology modelling using active state ß2-adrenergic receptor (ß2-AR). In addition, virtual screening and docking studies with both active and inactive state models reveal potential small molecule hits which could be considered as agonist-like and antagonist-like molecules. The results from the all-atom molecular dynamics simulations further confirmed that agonists and antagonists interact in different modes with the receptor.

Genome-wide survey of putative RNA-binding proteins encoded in the human proteome.

RNA-binding proteins (RBPs) are involved in various post-transcriptional gene regulatory processes and are also functionally important members of the ribosome and the spliceosome. However, RBPs and their interactions with RNA are less well-studied in comparison to DNA-binding proteins. We have classified the existing RBP structures, available in complexes with RNA and RNA/DNA hybrids, into different structural families and created Hidden Markov Models (HMMs). These structure-centric family HMMs, along with the sequence-centric family HMMs, were used as a primary database to systematically search the human proteome for the presence of putative RBPs. We have found more than 2600 gene products with RBP signatures in humans, of which around 28% are likely to bind to RNA but not DNA, whereas 9% might bind to both RNA and DNA. 11% of them do not contain an explicit functional annotation yet. Nearly 30% of the putative RBPs are exclusively nuclear, 15% have known disease associations and around 30% are enzymes. Around 40% of the proteins identified in this study are novel and have not been reported by recent large-scale studies on human RBPs.

PASS2 database for the structure-based sequence alignment of distantly related SCOP domain superfamilies: update to version 5 and added features.

Structure-based sequence alignment is an essential step in assessing and analysing the relationship of distantly related proteins. PASS2 is a database that records such alignments for protein domain superfamilies and has been constantly updated periodically. This update of the PASS2 version, named as PASS2.5, directly corresponds to the SCOPe 2.04 release. All SCOPe structural domains that share less than 40% sequence identity, as defined by the ASTRAL compendium of protein structures, are included. The current version includes 1977 superfamilies and has been assembled utilizing the structure-based sequence alignment protocol. Such an alignment is obtained initially through MATT, followed by a refinement through the COMPARER program. The JOY program has been used for structural annotations of such alignments. In this update, we have automated the protocol and focused on inclusion of new features such as mapping of GO terms, absolutely conserved residues among the domains in a superfamily and inclusion of PDBs, that are absent in SCOPe 2.04, using the HMM profiles from the alignments of the superfamily members and are provided as a separate list. We have also implemented a more user-friendly manner of data presentation and options for downloading more features. PASS2.5 version is available at http://caps.ncbs.res.in/pass2/.

Interactions Among Plant Transcription Factors Regulating Expression of Stress-responsive Genes.

Plants are simultaneously subjected to a variety of stress conditions in the field and are known to combat the hostile conditions by up/down-regulating number of genes. There exists a significant level of cross-talk between different stress responses in plants. In this study, we predict the interacting pairs of transcription factors that regulate the multiple abiotic stress-responsive genes in the plant Arabidopsis thaliana. We identified the interacting pair(s) of transcription factors (TFs) based on the spatial proximity of their binding sites. We also examined the interactions between the predicted pairs of TFs using molecular docking. Subsequent to docking, the best interaction pose was selected using our scoring scheme DockScore, which ranks the docked solutions based on several interface parameters and aims to find optimal interactions between proteins. We analyzed the selected docked pose for the interface residues and their conservation.

Population distribution analyses reveal a hierarchy of molecular players underlying parallel endocytic pathways.

Single-cell-resolved measurements reveal heterogeneous distributions of clathrin-dependent (CD) and -independent (CLIC/GEEC: CG) endocytic activity in Drosophila cell populations. dsRNA-mediated knockdown of core versus peripheral endocytic machinery induces strong changes in the mean, or subtle changes in the shapes of these distributions, respectively. By quantifying these subtle shape changes for 27 single-cell features which report on endocytic activity and cell morphology, we organize 1072 Drosophila genes into a tree-like hierarchy. We find that tree nodes contain gene sets enriched in functional classes and protein complexes, providing a portrait of core and peripheral control of CD and CG endocytosis. For 470 genes we obtain additional features from separate assays and classify them into early- or late-acting genes of the endocytic pathways. Detailed analyses of specific genes at intermediate levels of the tree suggest that Vacuolar ATPase and lysosomal genes involved in vacuolar biogenesis play an evolutionarily conserved role in CG endocytosis.

3PFDB+: improved search protocol and update for the identification of representatives of protein sequence domain families.

Protein domain families are usually classified on the basis of similarity of amino acid sequences. Selection of a single representative sequence for each family provides targets for structure determination or modeling and also enables fast sequence searches to associate new members to a family. Such a selection could be challenging since some of these domain families exhibit huge variation depending on the number of members in the family, the average family sequence length or the extent of sequence divergence within a family. We had earlier created 3PFDB database as a repository of best representative sequences, selected from each PFAM domain family on the basis of high coverage. In this study, we have improved the database using more efficient strategies for the initial generation of sequence profiles and implement two independent methods, FASSM and HMMER, for identifying family members. HMMER employs a global sequence similarity search, while FASSM relies on motif identification and matching. This improved and updated database, 3PFDB+ generated in this study, provides representative sequences and profiles for PFAM families, with 13 519 family representatives having more than 90% family coverage. The representative sequence is also highlighted in a two-dimensional plot, which reflects the relative divergence between family members. Representatives belonging to small families with short sequences are mainly associated with low coverage. The set of sequences not recognized by the family representative profiles, highlight several potential false or weak family associations in PFAM. Partial domains and fragments dominate such cases, along with sequences that are highly diverged or different from other family members. Some of these outliers were also predicted to have different secondary structure contents, which reflect different putative structure or functional roles for these domain sequences. Database URL: http://caps.ncbs.res.in/3pfdbplus/.