TranCEP: Predicting the substrate class of transmembrane transport proteins using compositional, evolutionary, and positional information

Transporters mediate the movement of compounds across the membranes that separate the cell from its environment and across the inner membranes surrounding cellular compartments. It is estimated that one third of a proteome consists of membrane proteins, and many of these are transport proteins. Given the increase in the number of genomes being sequenced, there is a need for computational tools that predict the substrates that are transported by the transmembrane transport proteins. In this paper, we present TranCEP, a predictor of the type of substrate transported by a transmembrane transport protein. TranCEP combines the traditional use of the amino acid composition of the protein, with evolutionary information captured in a multiple sequence alignment (MSA), and restriction to important positions of the alignment that play a role in determining the specificity of the protein. Our experimental results show that TranCEP significantly outperforms the state-of-the-art predictors. The results quantify the contribution made by each type of information used

Predicting Transporter Proteins and Their Substrate Specificity

The publication of numerous genome projects has resulted in an abundance of protein sequences, a significant number of which are still unannotated. Membrane proteins such as transporters, receptors, and enzymes are among the least characterized proteins due to their hydrophobic surfaces and lack of conformational stability. This research aims to build a proteome-wide system to determine transporter substrate specificity, which involves three phases: 1) distinguishing membrane proteins, 2) differentiating transporters from other functional types of membrane proteins, and 3) detecting the substrate specificity of the transporters. To distinguish membrane from non-membrane proteins, we propose a novel tool, TooT-M, that combines the predictions from transmembrane topology prediction tools and a selective set of classifiers where protein samples are represented by pseudo position-specific scoring matrix (Pse-PSSM) vectors. The results suggest that the proposed tool outperforms all state-of-the-art methods in terms of the overall accuracy and Matthews correlation coefficient (MCC). To distinguish transporters from other proteins, we propose an ensemble classifier, TooT-T, that is trained to optimally combine the predictions from homology annotation transfer and machine learning methods. The homology annotation transfer components detect transporters by searching against the transporter classification database (TCDB) using different thresholds. The machine learning methods include three models wherein the protein sequences are encoded using a novel encoding psi-composition. The results show that TooT-T outperforms all state-of-the-art de novo transporter predictors in terms of the overall accuracy and MCC. To detect the substrate specificity of a transporter, we propose a novel tool, TooT-SC, that combines compositional, evolutionary, and positional information to represent protein samples. TooT-SC can efficiently classify transport proteins into eleven classes according to their transported substrate, which is the highest number of predicted substrates offered by any de novo prediction tool. Our results indicate that TooT-SC significantly outperforms all of the state-of-the-art methods. Further analysis of the locations of the informative positions reveals that there are more statistically significant informative positions in the transmembrane segments (TMSs) than the non-TMSs, and there are more statistically significant informative positions that occur close to the TMSs compared to regions far from them

Changing the ligand-binding specificity of E. coli periplasmic binding protein RbsB by rational design and screening

Periplasmic binding proteins (PBPs) form a superfamily of bacterial proteins with a conserved bilobal structure, which are involved in substrate scavenging for bacterial cells. A wide variety of natural ligand-binding domains has evolved. PBPs are composed of two domains connected by a hinge region, which form a binding pocket between the two domains. They can be found in two stable conformations; in absence of ligand the PBP adopts an open conformation, where the binding pocket is exposed. In presence of the ligand, the protein changes to the closed conformation where the ligand is buried in the middle of the protein. This project focused on the ribose-binding protein of Escherichia coli (RbsB). Ribose binding to RbsB stabilizes the closed state. RbsB-bound ribose is presented to a cytoplasmic transport channels (RbsAC), from where it is imported into the cell, or interacts to membrane receptors (i.e., Trg) and can elicit a chemotactic signal. Due to their unique ligand-binding characteristics and wide variety of natural binding pockets PBPs have been of interest for the development of biosensors and bioreporter systems. PBP bioreporters were initiated over 20 years ago by a development in the group of Hazelbauer, who fused the C-terminal part of the E. coli EnvZ osmoregulation histidine kinase to the N-terminal part of the Trg methyl-accepting chemotaxis receptor protein, creating a hybrid receptor Trz1. Ligand bound galactose-binding protein (GBP) and ribose-bound RbsB interact with Trz1, which eventually leads to phosphorylation of the response regulator OmpR, activating transcription from the ompC promotor (and any reporter gene fused to this). In 2003, Hellinga’s group proposed that based on crystal structure information of ligand-bound PBPs variants with new ligand recognition specificities could be designed by computational approaches. Notably, they claimed the design of a RbsB-variant with nM affinity for recognition of 2,4,6-trinitrotoluene (TNT). This idea inspired the scientific community, because it could easily extend PBP-binding to a tremendous variety of compounds, including non- natural molecules, and would thus permit a wide variety of biosensor and bioreporter systems based on RbsB/GBP and Trz1. Unfortunately, independent engineering of some of the most promising published mutants failed to reproduce the reported in vivo and in vitro results. These studies further concluded that the published variants were actually misfolded proteins and/or impaired in stability as result of the introduced ligand-pocket mutations. This fact was largely ignored by Hellinga’s publications. Still inspired by the concept and trying to understand the reason of such limited success, our group raised the hypothesis that changing from ribose to TNT in a single step was likely unfeasible, but given the wide range of naturally evolved PBP ligand binding pockets, a step by step change of ribose binding to a non-natural analogue should be possible. To test this, we selected compounds with distinct differences but still chemically similar to ribose: 1,3-cyclohexanediol (13CHD) and cyclohexanol (CH). Mutant ligand binding pockets that might accommodate 13CHD and/or CH were computationally simulated and calculated using Rosetta, from which a list of critical amino acid residues to mutate in RbsB was selected. These were then synthesized and cloned into E. coli; a resulting set of 2 million mutants containing one of five possible substitutions at each of 9 selected critical amino acid positions. The library was introduced into an E. coli bioreporter strain, which carries the Trz1 hybrid signaling pathway coupled to GFP production when the (new) ligand would bind the (mutant) RbsB. The main goals of this work were to screen and characterize mutants from this first library, and potentially improve mutants for the new ligand binding in further rounds of mutagenesis. In the first part of this work a precise and user-friendly high-throughput strategy to screen the mutant library was developed. Clones were grown as individual microcolonies in alginate beads, to reduce single cell GFP expression variability, which were screened by fluorescence activated cell sorting (FACS) for gain-of-function GFP expression in presence of 13CHD. Six mutants with modest (1.5- fold) but consistent induction with 1 mM 13CHD were isolated. Moreover, these mutants completely lost the capacity to react to ribose. The RbsB mutants were characterized in terms of periplasmic space abundance, stability, secondary structure and ligand affinity. Isothermal microcalorimetry confirmed 13CHD binding, although only two mutants were sufficiently stable upon purification. Circular dichroism and quantification of periplasmic space abundance suggested the mutants to be prone to misfolding and/or defects in translocation. In the second part of this work, we used random and semi-random mutagenesis to improve the affinity and/or stability of the six isolated mutants with 13CHD binding capacity. Several mutant libraries were produced and screened with the previous described strategy. Variants displaying higher expression levels of GFP in presence of 13CHD were collected by FACS, and were used as starting point for the next round of evolution. This mutagenesis and rigorous screening strategy allowed us to isolate 7 mutants with improved (3.2-fold) GFP induction in presence of 13CHD and in a concentration- dependent manner. Several variants were observed that displayed open and closed conformations simultaneously, suggesting they were impaired in transition dynamics. Moreover, our screening strategy largely ignores potential variants with improved binding and closed conformation stability, but that are unable to interact with Trz1 receptor (e.i., trigger the signaling cascade). Finally in the third part of this work, we developed and tested an in vivo system to characterize the quality of the translocation process and receptor interactions. Wild-type- and mutant-RbsB proteins were fused to mCherry reporter protein to study protein abundance and subcellular localization. Whereas RbsB-mCherry proteins clearly localized to the periplasmic space and centered in polar regions depending on chemoreceptor availability, mutant-RbsB-mCherry expression resulted in high proportions of cells devoid of clear foci and low proportions of cells with multiple fluorescent foci, suggesting poorer translocation and mislocalisation. In addition, polar foci of mutants were less fluorescent, suggesting poorer chemoreceptor binding. By spiking further derivative mutant libraries generated by error-prone PCR without or with different proportions of E. coli expressing wild-type RbsB-mCherry we could estimate the potential improvement and deterioration of mutants with wild- type-like periplasmic localisation. The in vivo translocation system may thus be used to detect mutants with better signal transduction capacity. In conclusion, we firmly showed that design of PBP receptor proteins with new binding capacities for non-natural compounds is feasible, but still largely a matter of trial and error. The combination of computational simulations, random mutagenesis and rigorous screening allowed us to isolate variants with new recognition for 13CHD and loss of ribose binding. However, our results also showed that most predicted ligand-binding pocket mutations lead to poorly folding and functioning proteins, and it is likely that the dynamic transition needed between open and closed conformations of (here) RbsB is insufficiently understood and currently predictable to allow rational expansion to a wide range of new ligands. -- Les protéines de liaison périplasmiques (PLP) constituent une superfamille de protéines bactériennes avec une structure bilobée. Elles sont impliquées dans la captation de substrats pour les cellules bactériennes, et montrent grande diversité de domaines de liaison à des composés naturels. Les PLP sont composées de deux domaines connectés par une région charnière, ce qui forme une poche de liaison au substrat entre les deux domaines. Les PLP montrent deux états stables : ouverte en l’absence de ligand, conformation dans laquelle la poche de liaison est exposée, et fermée quand le ligand est séquestré dans la poche de liaison. Ce projet a porté sur l’étude de la PLP RbsB liant le ribose chez Escherichia coli. La liaison du ribose stabilise l’état fermé de RbsB et permet l’interaction avec le transporteur cytoplasmique RbsAC et son passage dans le cytoplasme de la cellule, ou son interaction avec des récepteurs membranaires tels que Trg permettant en une réponse chimiotactique. Étant données leurs caractéristiques uniques de liaison aux ligands et la grande variété de poches de liaison naturellement observée chez les PLP, elles présentent un grand intérêt pour le développement de biosenseurs et de systèmes biorapporteurs. Les premiers biorapporteurs basés sur des PLP ont été développés 20 ans auparavant par le groupe de Hazelbauer. Cette équipe a fusionné la partie C-terminale de la protéine kinase à histidine impliquée dans l’osmorégulation (EnvZ) et l’extrémité N-terminale du récepteur chimiotactique accepteur de groupement méthyle (Trg), pour créer le récepteur hybride Trz1. Les PLP liant le galactose (GBP) et le ribose (RbsB) interagissent avec Trz1, ce qui entraine la phosphorylation du régulateur réponse OmpR qui lui-même va activer la transcription à partir du promoteur du gène ompC (ou n’importe quel système rapporteur placé en aval de ce promoteur). En 2003, le groupe de Hellinga proposait que, sur la base de la structure cristallographique de différents PLP liées à leur ligand, des variants reconnaissant de nouveaux ligands pourraient être générés sur la base d’une approche informatique. En particulier, cette équipe se targue d’avoir générer un variant de RbsB permettant de lier le 2,4,6-trinitrotoluène (TNT) avec une affinité de l’ordre du nanomolaire. Cette idée a inspiré la communauté scientifique car cette approche pourrait s’étendre à une diversité incroyable de composés naturels ou non, ce qui permettrait le développement de biosenseurs et biorapporteurs variés basés sur ce système. Malheureusement, la construction des mutants les plus prometteurs par des équipes indépendantes n’ont pas permis de rapporter de l’activité in vivo et/ou in vitro. Cela a été ignoré dans les publications du groupe Hellinga. Inspirés par ce concept et voulant savoir quelles étaient les raisons de ce succès quelque peu limité, notre groupe a émis l’hypothèse que le changement de spécificité de RbsB du ribose au TNT en une étape était probablement infaisable mais, étant donnée la grande diversité de poches de liaisons naturellement observées chez les LPL, un changement pas à pas du ribose vers un composé analogue non naturel devrait être possible. Pour tester cela, nous avons sélectionné des composés distincts du ribose mais présentant tout de même des similarités : 1,3-cyclohexanediol (13CHD) and cyclohexanol (CH). Des mutants qui pourraient accueillir le 13CHD et/ou CH ont été générés par simulation informatique en utilisant le programme Rosetta, lequel a fourni une liste d’acides aminés critiques à muter. Une librairie de mutant a été synthétisée, celle-ci contenant 2 millions de variants de RbsB avec 1 substition parmi 5 possibles à 9 positions sélectionnées pour leur aspect critique dans la reconnaissance du substrat. La librairie a été introduite et criblée chez une souche reportrice d’E. coli contenant la chaine de signalisation hybride Trz1 couplée à la production de la protéine fluorescente verte (GFP) lorsque le (nouveau) ligand se liera à la protéine RbsB (sauvage ou mutante). Le but principal de ce travail était de caractériser cette librairie de mutants, et éventuellement d’améliorer la capacité de ces mutants à lier un autre composant par des cycles de mutagénèses additionnels. Dans la première partie de ce travail, une stratégie simple et efficace pour cribler la librairie de mutant a été développée. Les différents clones/variants ont été cultivés individuellement en microcolonies dans des billes d’alginate afin de réduire la variabilité du signal GFP observé au niveau de la cellule unique. Les billes ont été analysées par trieur de cellules reposant sur la fluorescence (FACS) afin de détecter des mutants présentant une activité GFP accrue en présence de 13CHD. Six mutants ont été isolés pour leur modeste mais significative induction (1,5 fois) en présence de 1 mM de 13CHD. De plus, ces mutants avaient totalement perdu leur capacité à réagir au ribose. Les mutants RbsB ont été caractérisés plus en détails pour leur localisation dans périplasme, leur stabilité, leur abondance et leur affinité pour le ligand. La technique de microcalorimétrie isotherme a confirmé que ces mutants lient le 13CHD, bien que seulement 2 de ces protéines mutantes se soient révélées suffisamment stables après purification. L’analyse par dichroïsme circulaire et la quantification de l’abondance des protéines dans l’espace périplasmique suggèrent que les protéines mutantes sont sujettes à un mauvais repliement et/ou un problème dans la translocation du cytoplasme au périplasme. Dans une seconde partie, nous avons muté les six mutants isolés précédemment de façon aléatoire ou semi-aléatoire afin d’améliorer leur affinité pour le 13CHD et/ou leur stabilité. Plusieurs librairies de mutants ont été produites et analysées selon la méthode décrite plus tôt. Les variants montrant une plus forte expression du système rapporteur GFP en présence de 13CHD ont été isolés par FACS, et utilisés comme point de départ pour la prochaine étape d’évolution. Cette mutagénèse et l’analyse rigoureuse des librairies nous ont permis d’isoler 7 mutants avec une augmentation de 3,2 fois du signal GFP en présence de 13CHD, et d’une façon dose-dépendante. Plusieurs variants ont montré qu’ils adoptaient la conformation ouverte et fermées au sein de la population bactérienne. Cette dernière observation suggère que ces mutants sont affectés dans leur capacité à passer d’une conformation à l’autre. De plus, notre stratégie de crible ne tient pas compte les variants qui montreraient une liaison accrue et une bonne stabilité de la conformation fermée, mais qui seraient incapables d’interagir avec le récepteur Trz1 (et donc de déclencher la cascade de signalisation du rapporteur). Finalement, dans la troisième partie de ce travail, nous avons développé et testé un système in vivo permettant de caractériser la qualité du processus de translocation dans l’espace périplasmique et l’interaction avec les récepteurs. Les protéines RbsB sauvage et mutantes ont été fusionnées à la protéine fluorescente rouge mCherry afin de visualiser l’abondance et la localisation sub-cellulaire des protéines au niveau de la cellule unique en utilisant la microscopy à épifluorescence et le traitement des images obtenues. Alors que la protéine de fusion RbsB sauvage montre une localisation périplasmique centrées au niveau des pôles de la cellule dépendamment de la disponibilité des chimiorécepteurs, les fusions avec les variants de RbsB montraient une forte proportion de cellules dépourvues de foci, et une faible proportion de cellules avec de multiples foci, suggérant une plus faible liaison aux chimiorécepteurs. En analysant plus en détails des librairies de mutants générées par PCR mutagène, en mélangeant ou non avec des cellules contenant la protéine de fusion RbsB sauvage, nous avons pu estimer l’amélioration potentielle ou la détérioration des qualités des mutants RbsB par rapport au sauvage en terme de localisation périplasmique. Ce système de translocation in vivo pourrait être utilisé afin de détecter des mutants permettant une meilleure transduction du signal. En conclusion, nous avons montré que la conception de protéines réceptrices PLP présentant de nouvelles capacités de liaison pour des composés non naturels est bien faisable, mais repose encore sur une stratégie d’essais et erreurs. La combinaison de simulations informatiques, de mutagénèses aléatoires et de crible rigoureux nous a permis d’isoler des variants de RbsB avec une capacité à reconnaitre le 13CHD, tout en ne liant plus le ribose. Néanmoins, nos résultats ont également montré que la plupart des prédictions de mutations au niveau de la poche de liaison ont mené à un mauvais repliement ou fonctionnement des protéines. Il est très probable que la dynamique de transition entre la conformation ouverte et fermée (de RbsB pour cette étude) ne soit pas encore assez bien comprise, et donc actuellement non prédictable pour permettre le test d’une grande variété de nouveaux ligands

BOOST THE DSICOVERY OF MRP7/ABCC10 SUBSTRATES AND INHIBITORS: ESTABLISHMENT OF NEW IN VITRO AND IN SILICO MODELS

ATP-binding cassette (ABC) transporters are responsible for the efflux of structurally distinct endo- and xenobiotics energized by ATP hydrolysis. MRP7/ABCC10 belongs to the 10th member of subfamily C and responsible for mediating MDR against a series of chemotherapeutic drugs such as taxanes, epothilones, Vinca alkaloids, anthracyclines and epipodophyllotoxins. Establishment of new in silico and in vitro models for MRP7 substrates/inhibitors prediction Considering the limited knowledge of MRP7, we established a homology model based on bovine MRP1 cryo-EM models. The final model was used for protein global motion analysis and docking analysis. Before docking, potential drug binding pockets were identified and evaluated. Next, MRP7 substrates and inhibitors were docked into drug binding pockets. We found that docked inhibitors and substrates formed separate clusters, from which a substrate binding region and an inhibitor binding region were proposed. This homology protein model enables the docking analysis of potential MRP7 ligands for future studies. Moreover, we established a new SKOV3/MRP7 cell line which exhibits similar drug resistance profile as the previously established HEK/MRP7 cell line. This new cell line is valuable for MRP7 substrates and inhibitors discovery. Last but not the least, we established a novel machine learning model named Mrp7Pred for large-scale MRP7 substrates/inhibitors prediction. The model was also deployed as a web server and is freely available to users in http://www.mrp7pred.com. We successfully identified 2 substrates and 4 inhibitors from 70 FDA-approved drugs using Mrp7Pred. New synthetic agents targeting MRP7 and overcomes MRP7-medited MDR Previously, we identified two synthetic compounds, CMP25 and CP55, as potent ABCB1 and ABCG2 inhibitors. Here we found these two compounds also significantly reversed the MDR mediated by MRP7. Both compounds significantly sensitized MRP7- overexpressing HEK/MRP7 cells to paclitaxel and vincristine. Western blotting indicates that neither CMP25 nor CP55 alters MRP7 expression level. Immunofluorescence showed that the subcellular localization of MRP7 was not altered by these two compounds. However, intracellular accumulation of [3H]-paclitaxel and [3H]-vincristine were significantly increased while the efflux was significantly reduced when co- administered with CMP25 or CP55. Hydrophobic interactions were predicted as the major contributors in stabilizing the drug-protein complex via docking analysis

In Silico prediction of the Caspase degradome

Ph.DDOCTOR OF PHILOSOPH

Harnessing the evolutionary information on oxygen binding proteins through Support Vector Machines based modules

© The Author(s) 2018 This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Abstract Objectives The arrival of free oxygen on the globe, aerobic life is becoming possible. However, it has become very clear that the oxygen binding proteins are widespread in the biosphere and are found in all groups of organisms, including prokaryotes, eukaryotes as well as in fungi, plants, and animals. The exponential growth and availability of fresh annotated protein sequences in the databases motivated us to develop an improved version of “Oxypred” for identifying oxygen-binding proteins. Results In this study, we have proposed a method for identifying oxy-proteins with two different sequence similarity cutoffs 50 and 90%. A different amino acid composition based Support Vector Machines models was developed, including the evolutionary profiles in the form position-specific scoring matrix (PSSM). The fivefold cross-validation techniques were applied to evaluate the prediction performance. Also, we compared with existing methods, which shows nearly 97% recognition, but, our newly developed models were able to recognize almost 99.99 and 100% in both oxy-50 and 90% similarity models respectively. Our result shows that our approaches are faster and achieve a better prediction performance over the existing methods. The web-server Oxypred2 was developed for an alternative method for identifying oxy-proteins with more additional modules including PSSM, available at http://bioinfo.imtech.res.in/servers/muthu/oxypred2/home.html

In vivo and in vitro characterization of Staphylococcus aureus and Bacillus subtilis polyglycerolphosphate lipoteichoic acid synthases

Staphylococcus aureus lipoteichoic acid (LTA) consists of a 1,3-linked polyglycerolphosphate chain retained in the bacterial membrane by a glycolipid anchor. The LTA backbone is produced by the lipoteichoic acid synthase LtaS, a membrane protein with five transmembrane helices and a large extracellular enzymatic domain (eLtaS). Proteomic studies revealed that LtaS is efficiently cleaved, and here it was demonstrated that the eLtaS domain is released into the culture supernatant as well as partially retained within the cell wall fraction. However, using an in vivo LtaS activity assay, it was shown that only the full-length LtaS enzyme is able to synthesize LTA. Neither expression of a secreted eLtaS variant, created by replacing the N-terminal membrane domain with a conventional signal sequence, nor expression of eLtaS fused to a single or multi-transmembrane domains of other staphylococcal proteins resulted in the production of LTA. These data indicate that the transmembrane domain of LtaS play an essential, yet unknown, role in LtaS enzyme function. In addition, the protease responsible for LtaS cleavage was identified. It was found that a S. aureus strain in which the gene encoding for the essential signal peptidase SpsB was cloned under inducible expression control showed an accumulation of the full-length LtaS enzyme in the absence of the inducer. These data suggest that SpsB is involved in LtaS cleavage. Four LtaS orthologues, YflE, YfnI, YqgS and YvgJ, are present in Bacillus subtilis. Using an in vitro enzyme assay and purified protein, it was determined that all four B. subtilis proteins are Mn2+-dependent metal enzymes that use the lipid phosphatidylglycerol as substrate. It was shown that YflE, YfnI and YqgS are bonafide LTA synthases capable of producing polyglycerolphosphate chains, while YvgJ appears to function as an LTA primase, as indicated by the accumulation of a glycolipid with the expected chromatographic mobility of GroP-Glc2-DAG. Taken together, experimental evidence for the enzyme function of all four B. subtilis LtaStype proteins is provided in this work and it was shown that all four enzymes are involved in the LTA synthesis process