GrAfSS: a webserver for substructure similarity searching and comparisons in the structures of proteins and RNA.

The GrAfSS (Graph theoretical Applications for Substructure Searching) webserver is a platform to search for three-dimensional substructures of: (i) amino acid side chains in protein structures; and (ii) base arrangements in RNA structures. The webserver interfaces the functions of five different graph theoretical algorithms - ASSAM, SPRITE, IMAAAGINE, NASSAM and COGNAC - into a single substructure searching suite. Users will be able to identify whether a three-dimensional (3D) arrangement of interest, such as a ligand binding site or 3D motif, observed in a protein or RNA structure can be found in other structures available in the Protein Data Bank (PDB). The webserver also allows users to determine whether a protein or RNA structure of interest contains substructural arrangements that are similar to known motifs or 3D arrangements. These capabilities allow for the functional annotation of new structures that were either experimentally determined or computationally generated (such as the coordinates generated by AlphaFold2) and can provide further insights into the diversity or conservation of functional mechanisms of structures in the PDB. The computed substructural superpositions are visualized using integrated NGL viewers. The GrAfSS server is available at http://mfrlab.org/grafss/.

Graph Theoretical Methods and Workflows for Searching and Annotation of RNA Tertiary Base Motifs and Substructures.

The increasing number and complexity of structures containing RNA chains in the Protein Data Bank (PDB) have led to the need for automated structure annotation methods to replace or complement expert visual curation. This is especially true when searching for tertiary base motifs and substructures. Such base arrangements and motifs have diverse roles that range from contributions to structural stability to more direct involvement in the molecule's functions, such as the sites for ligand binding and catalytic activity. We review the utility of computational approaches in annotating RNA tertiary base motifs in a dataset of PDB structures, particularly the use of graph theoretical algorithms that can search for such base motifs and annotate them or find and annotate clusters of hydrogen-bond-connected bases. We also demonstrate how such graph theoretical algorithms can be integrated into a workflow that allows for functional analysis and comparisons of base arrangements and sub-structures, such as those involved in ligand binding. The capacity to carry out such automatic curations has led to the discovery of novel motifs and can give new context to known motifs as well as enable the rapid compilation of RNA 3D motifs into a database.

Regulation of Glycine Cleavage and Detoxification by a Highly Conserved Glycine Riboswitch in Burkholderia spp.

The glycine riboswitch is a known regulatory element that is unique in having two aptamers that are joined by a linker region. In this study, we investigated a glycine riboswitch located in the 5' untranslated region of a glycine cleavage system homolog (gcvTHP) in Burkholderia spp. Structure prediction using the sequence generated a model with a glycine binding pocket composed of base-triple interactions (G62-A64-A86 and G65-U84-C85) that are supported by A/G minor interactions (A17-C60-G88 and G16-C61-G87, respectively) and two ribose-zipper motifs (C11-G12 interacting with A248-A247 and C153-U154 interacting with A79-A78) which had not been previously reported. The capacity of the riboswitch to bind to glycine was experimentally validated by native gel assays and the crucial role of interactions that make up the glycine binding pocket were proven by mutations of A17U and G16C which resulted in conformational differences that may lead to dysfunction. Using glycine supplemented minimal media, we were able to prove that the expression of the gcvTHP genes found downstream of the riboswitch responded to the glycine concentrations introduced thus confirming the role of this highly conserved Burkholderia riboswitch and its associated genes as a putative glycine detoxification system in Burkholderia spp.

Side chain similarity comparisons for integrated drug repositioning and potential toxicity assessments in epidemic response scenarios: The case for COVID-19.

Structures of protein-drug-complexes provide an atomic level profile of drug-target interactions. In this work, the three-dimensional arrangements of amino acid side chains in known drug binding sites (substructures) were used to search for similarly arranged sites in SARS-CoV-2 protein structures in the Protein Data Bank for the potential repositioning of approved compounds. We were able to identify 22 target sites for the repositioning of 16 approved drug compounds as potential therapeutics for COVID-19. Using the same approach, we were also able to investigate the potentially promiscuous binding of the 16 compounds to off-target sites that could be implicated in toxicity and side effects that had not been provided by any previous studies. The investigations of binding properties in disease-related proteins derived from the comparison of amino acid substructure arrangements allows for effective mechanism driven decision making to rank and select only the compounds with the highest potential for success and safety to be prioritized for clinical trials or treatments. The intention of this work is not to explicitly identify candidate compounds but to present how an integrated drug repositioning and potential toxicity pipeline using side chain similarity searching algorithms are of great utility in epidemic scenarios involving novel pathogens. In the case of the COVID-19 pandemic caused by the SARS-CoV-2 virus, we demonstrate that the pipeline can identify candidate compounds quickly and sustainably in combination with associated risk factors derived from the analysis of potential off-target site binding by the compounds to be repurposed.

Phylogenetic comparison between Type IX Secretion System (T9SS) protein components suggests evidence of horizontal gene transfer.

Porphyromonas gingivalis is one of the major bacteria that causes periodontitis. Chronic periodontitis is a severe form of periodontal disease that ultimately leads to tooth loss. Virulence factors that contribute to periodontitis are secreted by Type IX Secretion System (T9SS). There are aspects of T9SS protein components that have yet to be characterised. Thus, the aim of this study is to investigate the phylogenetic relationship between members of 20 T9SS component protein families. The Bayesian Inference (BI) trees for 19 T9SS protein components exhibit monophyletic clades for all major classes under Bacteroidetes with strong support for the monophyletic clades or its subclades that is consistent with phylogeny exhibited by the constructed BI tree of 16S rRNA. The BI tree of PorR is different from the 19 BI trees of T9SS protein components as it does not exhibit monophyletic clades for all major classes under Bacteroidetes. There is strong support for the phylogeny exhibited by the BI tree of PorR which deviates from the phylogeny based on 16S rRNA. Hence, it is possible that the porR gene is subjected to horizontal transfer as it is known that virulence factor genes could be horizontally transferred. Seven genes (porR included) that are involved in the biosynthesis of A-LPS are found to be flanked by insertion sequences (IS5 family transposons). Therefore, the intervening DNA segment that contains the porR gene might be transposed and subjected to conjugative transfer. Thus, the seven genes can be co-transferred via horizontal gene transfer. The BI tree of UgdA does not exhibit monophyletic clades for all major classes under Bacteroidetes which is similar to the BI tree of PorR (both are a part of the seven genes). Both BI trees also exhibit similar topology as the four identified clusters with strong support and have similar relative positions to each other in both BI trees. This reinforces the possibility that porR and the other six genes might be horizontally transferred. Other than the BI tree of PorR, the 19 other BI trees of T9SS protein components also exhibit evidence of horizontal gene transfer. However, their genes might undergo horizontal gene transfer less frequently compared to porR because the intervening DNA segment that contains porR is easily exchanged between bacteria under Bacteroidetes due to the presence of insertion sequences (IS5 family transposons) that flank it. In conclusion, this study can provide a better understanding about the phylogeny of T9SS protein components.