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Co-evolving pair of residues predicted by CMAT. The N-terminal domain corresponds to residues 1 to 153, while residues 154â 349 make up the TIR domain in the concatenated multiple sequence alignment. (DOCX 16Â kb)

Appendix S1. List of organisms used for co-evolution analysis (DOCX 16Â kb)

Vibrio parahaemolyticus is one of the leading causative agents of foodborne diseases in humans. In this study, the proteome profiles of the pandemic strain V. parahaemolyticus SC192 belonging to the O3:K6 serovar during the planktonic and biofilm stages were analyzed by two-dimensional liquid chromatography coupled to tandem mass spectrometry. This non-gel-based multidimensional protein identification technology approach identified 45.5% of the proteome in the reference genome V. parahaemolyticus RIMD 2210633. This is the largest proteome coverage obtained so far in V. parahaemolyticus and provides evidence for expression of 27% of the hypothetical proteins. Comparison of the planktonic and biofilm proteomes based on their cluster of orthologous groups, gene ontologies and KEGG pathways provides basic information on biofilm specific functions and pathways. To the authors' knowledge, this is the first study to generate a global proteome profile of the pandemic strain of V. parahaemolyticus and the method reported here could be used to rapidly obtain a snapshot of the proteome of any microorganism at a given condition.

List of proteins used in the resubstitution test. (DOC 102 kb)

Lesk-Hubbard plot of the multiple structural alignment of the homology models. (PDF 40 kb)

Family-specific list of RNA that bind to protein chains belonging to the family. (DOC 1776 kb)

Cladograms of LuxS protein sequences showing (A) taxonomic distribution of species (Colour code: Blue: Firmicutes; Pink: Actinobacteria; Yellow: Bacteroidetes; Cyan: Spirochaetes; Red: Proteobacteria; Brown: Fusobacteria; Green: Deinococcus-Thermales) and habitat distribution of bacteria possessing the LuxS proteins (B), colour codes: Pink: Gut/Faecal microorganisms; Purple: Animal and Human pathogens; Brown: Soil-dwelling bacteria; Yellow-green: Food-associated bacteria; Forest-green: Plant-associated bacteria; Sky blue: Halophiles; Navy blue: Alkaliphiles; Cyan: Psychrophiles; Orange: Thermophiles; Grey: Radiation-resistant bacteria; Yellow: Hydrothermal vent inhabiting bacteria; Light blue: Marine bacteria; Blue-green: Plant pathogens; Black: Habitat information not available). (PDF 2242 kb)

Phylogenetic tree of LuxS sequences showing habitat distribution of sequences. Colour codes: Pink: Gut/Faecal microorganisms; Purple: Animal and Human pathogens; Brown: Soil-dwelling bacteria; Yellow-green: Food-associated bacteria; Forest-green: Plant-associated bacteria; Sky blue: Halophiles; Navy blue: Alkaliphiles; Cyan: Psychrophiles; Orange: Thermophiles; Grey: Radiation-resistant bacteria; Yellow: Hydrothermal vent inhabiting bacteria; Lignt blue: Marine bacteria; Blue-green: Plant pathogens; Black: Habitat information not available (PDF 108 kb)

Details of proteins used as the negative test set. (DOC 150 kb)

Text A. List of scaffolds as marked from START to END in Fig. 4. Text B. List of IDs of transcripts more abundant in Krishna as compared to Rama subtype (from top to bottom) marked in Fig. 5a. Text C. List of IDs of transcripts more abundant in Rama as compared to Krishna subtype (from top to bottom) as marked in Fig. 5b. Text D. Multiple sequence alignment of amyrin synthases with hits in Tulsi genome.

Presence of essential genes in O.tenuiflorum (Tulsi) at three levels; a) in only paired end assembly (ab-initio gene prediction), b) in paired end and mate-pair assemblyâ s Level 2 [evidence from RNAseq, EST and known tulsi genes], c) in paired end and mate-paired assemblyâ s Level 1 (gene prediction).

Repeat elements identified in Tulsi genome assembly and classified in different groups of repeats.

Pathways of all the 14 important medicinal metabolites of the Tulsi genome which were studied in detail.

Statistics of scaffold length comparison from assemblies of PE and MPâ +â PE together.

Completeness of assembly and presence of essential genes by CEGMA results for O. tenuiflorum at two levels; (a) only in PE assembly (b) in PEâ +â PM assembly.

Precise functioning and fine-tuning of Toll-like receptor 4 (TLR4) signaling is a critical requirement for the smooth functioning of the innate immune system, since aberrant TLR4 activation causes excessive production of pro-inflammatory cytokines and interferons. This can result in life threatening conditions such as septic shock and other inflammatory disorders. The TRIF-related adaptor molecule (TRAM) adaptor protein is unique to the TLR4 signaling pathway and abrogation of TRAM-mediated TLR4 signaling is a promising strategy for developing therapeutics aimed at disrupting TRAM interactions with other components of the TLR4 signaling complex. The VIPER motif from the vaccinia virus-producing protein, A46 has been reported to disrupt TRAM-TLR4 interactions. We have exploited this information, in combination with homology modelling and docking approaches, to identify a potential binding site on TRAM lined by the BB loop and αC helix. Virtual screening of commercially available small molecules targeting the binding site enabled to short-list 12 small molecules to abrogate TRAM-mediated TLR4 signaling. Molecular dynamics and molecular mechanics calculations have been performed for the analysis of these receptor-ligand interactions.

Sequences of putative terpene synthases in O. tenuiflorum genome.

Scaffold lengths distribution in the MPâ +â PE and PE assemblies.

Analysis of structural variation in domain superfamilies can reveal constraints in protein evolution which aids protein structure prediction and classification. Structure-based sequence alignment of distantly related proteins, organized in PASS2 database, provides clues about structurally conserved regions among different functional families. Some superfamily members show large structural differences which are functionally relevant. This paper analyses the impact of structural divergence on function for multi-member superfamilies, selected from the PASS2 superfamily alignment database. Functional annotations within superfamilies, with structural outliers or ‘rebels’, are discussed in the context of structural variations. Overall, these data reinforce the idea that functional similarities cannot be extrapolated from mere structural conservation. The implication for fold-function prediction is that the functional annotations can only be inherited with very careful consideration, especially at low sequence identities.

Proteins may be related to each other very specifically as homologous subfamilies. Proteins can also be related to diverse proteins at the super family level. It has become highly important to characterize the existing sequence databases by their signatures to facilitate the function annotation of newly added sequences. The algorithm described here uses a scheme for the classification of odorant binding proteins on the basis of functional residues and Cys-pairing. The cysteine-based scoring scheme not only helps in unambiguously identifying families like odorant binding proteins (OBPs), but also aids in their classification at the subfamily level with reliable accuracy. The algorithm was also applied to yet another cysteine-rich family, where similar accuracy was observed that ensures the application of the protocol to other families.

UNC-104/KIF1A is a Kinesin-3 motor that transports synaptic vesicles from the cell body towards the synapse by binding to PI(4,5)P2 through its PH domain. The fate of the motor upon reaching the synapse is not known. We found that wild-type UNC-104 is degraded at synaptic regions through the ubiquitin pathway and is not retrogradely transported back to the cell body. As a possible means to regulate the motor, we tested the effect of cargo binding on UNC-104 levels. The unc-104(e1265) allele carries a point mutation (D1497N) in the PI(4,5)P2 binding pocket of the PH domain, resulting in greatly reduced preferential binding to PI(4,5)P2 in vitro and presence of very few motors on pre-synaptic vesicles in vivo. unc-104(e1265) animals have poor locomotion irrespective of in vivo PI(4,5)P2 levels due to reduced anterograde transport. Moreover, they show highly reduced levels of UNC-104 in vivo. To confirm that loss of cargo binding specificity reduces motor levels, we isolated two intragenic suppressors with compensatory mutations within the PH domain. These show partial restoration of in vitro preferential PI(4,5)P2 binding and presence of more motors on pre-synaptic vesicles in vivo. These animals show improved locomotion dependent on in vivo PI(4,5)P2 levels, increased anterograde transport, and partial restoration of UNC-104 protein levels in vivo. For further proof, we mutated a conserved residue in one suppressor background. The PH domain in this triple mutant lacked in vitro PI(4,5)P2 binding specificity, and the animals again showed locomotory defects and reduced motor levels. All allelic variants show increased UNC-104 levels upon blocking the ubiquitin pathway. These data show that inability to bind cargo can target motors for degradation. In view of the observed degradation of the motor in synaptic regions, this further suggests that UNC-104 may get degraded at synapses upon release of cargo., Author Summary The cell body and the synapse in a neuron are often separated by significant distance, which is spanned by the axon connecting the two. Transport of various cargoes along the axonal highway is very important for neuronal function. The regulation of this complex process is not well understood. Using the Caenorhabditis elegans model system, we have demonstrated for the first time the fate of a motor after it carries its cargo to the synapse from the cell body. We show that the UNC-104 motor, which carries pre-synaptic vesicles to the synapse, is degraded once it gets there. Moreover, our genetic studies show evidence that loss of cargo binding targets the motor for degradation, suggesting an attractive mechanism for the regulation of motors at the synapse. Our study opens up several further questions, such as the mechanism of motor degradation, and has significant implications for regulation of cargo transport.

International audience; no abstract