Functional discrimination of membrane proteins using machine learning techniques

<p>Abstract</p> <p>Background</p> <p>Discriminating membrane proteins based on their functions is an important task in genome annotation. In this work, we have analyzed the characteristic features of amino acid residues in membrane proteins that perform major functions, such as channels/pores, electrochemical potential-driven transporters and primary active transporters.</p> <p>Results</p> <p>We observed that the residues Asp, Asn and Tyr are dominant in channels/pores whereas the composition of hydrophobic residues, Phe, Gly, Ile, Leu and Val is high in electrochemical potential-driven transporters. The composition of all the amino acids in primary active transporters lies in between other two classes of proteins. We have utilized different machine learning algorithms, such as, Bayes rule, Logistic function, Neural network, Support vector machine, Decision tree etc. for discriminating these classes of proteins. We observed that most of the algorithms have discriminated them with similar accuracy. The neural network method discriminated the channels/pores, electrochemical potential-driven transporters and active transporters with the 5-fold cross validation accuracy of 64% in a data set of 1718 membrane proteins. The application of amino acid occurrence improved the overall accuracy to 68%. In addition, we have discriminated transporters from other α-helical and β-barrel membrane proteins with the accuracy of 85% using k-nearest neighbor method. The classification of transporters and all other proteins (globular and membrane) showed the accuracy of 82%.</p> <p>Conclusion</p> <p>The performance of discrimination with amino acid occurrence is better than that with amino acid composition. We suggest that this method could be effectively used to discriminate transporters from all other globular and membrane proteins, and classify them into channels/pores, electrochemical and active transporters.</p

Phylogenomics of Reichenowia parasitica, an Alphaproteobacterial Endosymbiont of the Freshwater Leech Placobdella parasitica

Although several commensal alphaproteobacteria form close relationships with plant hosts where they aid in (e.g.,) nitrogen fixation and nodulation, only a few inhabit animal hosts. Among these, Reichenowia picta, R. ornata and R. parasitica, are currently the only known mutualistic, alphaproteobacterial endosymbionts to inhabit leeches. These bacteria are harbored in the epithelial cells of the mycetomal structures of their freshwater leech hosts, Placobdella spp., and these structures have no other obvious function than housing bacterial symbionts. However, the function of the bacterial symbionts has remained unclear. Here, we focused both on exploring the genomic makeup of R. parasitica and on performing a robust phylogenetic analysis, based on more data than previous hypotheses, to test its position among related bacteria. We sequenced a combined pool of host and symbiont DNA from 36 pairs of mycetomes and performed an in silico separation of the different DNA pools through subtractive scaffolding. The bacterial contigs were compared to 50 annotated bacterial genomes and the genome of the freshwater leech Helobdella robusta using a BLASTn protocol. Further, amino acid sequences inferred from the contigs were used as queries against the 50 bacterial genomes to establish orthology. A total of 358 orthologous genes were used for the phylogenetic analyses. In part, results suggest that R. parasitica possesses genes coding for proteins related to nitrogen fixation, iron/vitamin B translocation and plasmid survival. Our results also indicate that R. parasitica interacts with its host in part by transmembrane signaling and that several of its genes show orthology across Rhizobiaceae. The phylogenetic analyses support the nesting of R. parasitica within the Rhizobiaceae, as sister to a group containing Agrobacterium and Rhizobium species

A Novel Protein Kinase-Like Domain in a Selenoprotein, Widespread in the Tree of Life

Selenoproteins serve important functions in many organisms, usually providing essential oxidoreductase enzymatic activity, often for defense against toxic xenobiotic substances. Most eukaryotic genomes possess a small number of these proteins, usually not more than 20. Selenoproteins belong to various structural classes, often related to oxidoreductase function, yet a few of them are completely uncharacterised

In vitro functional characterization of BtuCD-F, the Escherichia coli ABC transporter for vitamin B12 uptake

BtuCD is an ATP binding cassette (ABC) transporter that facilitates uptake of vitamin B-12 into the cytoplasm of Escherichia coli. The crystal structures of BtuCD and its cognate periplasmic binding protein BtuF have been recently determined. We have now explored BtuCD-F function in vitro, both in proteoliposomes and in various detergents. BtuCD reconstituted into proteoliposomes has a significant basal ATP hydrolysis rate that is stimulated by addition of BtuF and inhibited by sodium ortho-vanadate. When using different detergents to solubilize BtuCD, the basal ATP hydrolysis rate, the ability of BtuF to stimulate hydrolysis, and the extent to which sodium ortho-vanadate inhibits ATP hycrolysis all vary significantly. Reconstituted BtuCD can mediate transport of vitamin B12 against a concentration gradient when coupled to ATP hydrolysis by BtuD in the liposome lumen and BtuF outside the liposomes. These in vitro studies establish the functional competence of the BtuCD and BtuF preparations used in the crystallographic analyses for both ATPase and transport activities. Furthermore, the tight binding of BtuF to BtuCD under the conditions studied suggests that the binding protein may not dissociate from the transporter during the catalytic cycle, which may be relevant to the mechanisms of other ABC transporter systems

Conformational Change of a Tryptophan Residue in BtuF Facilitates Binding and Transport of Cobinamide by the Vitamin B12 Transporter BtuCD-F

BtuCD-F is an ABC transporter that mediates cobalamin uptake into Escherichia coli. Early in vivo data suggested that BtuCD-F might also be involved in the uptake of cobinamide, a cobalamin precursor. However, neither was it demonstrated that BtuCD-F indeed transports cobinamide, nor was the structural basis of its recognition known. We synthesized radiolabeled cyano-cobinamide and demonstrated BtuCD-catalyzed in vitro transport, which was ATP- and BtuF-dependent. The crystal structure of cobinamide-bound BtuF revealed a conformational change of a tryptophan residue (W66) in the substrate binding cleft compared to the structure of cobalamin-bound BtuF. High-affinity binding of cobinamide was dependent on W66, because mutation to most other amino acids substantially reduced binding. The structures of three BtuF W66 mutants revealed that tight packing against bound cobinamide was only provided by tryptophan and phenylalanine, in line with the observed binding affinities. In vitro transport rates of cobinamide and cobalamin were not influenced by the substitutions of BtuF W66 under the experimental conditions, indicating that W66 has no critical role in the transport reaction. Our data present the molecular basis of the cobinamide versus cobalamin specificity of BtuCD-F and provide tools for in vitro cobinamide transport and binding assays

Structural basis of nanobody-mediated blocking of BtuF, the cognate substrate-binding protein of the Escherichia coli vitamin B12 transporter BtuCD

Abstract Bacterial ABC importers catalyze the uptake of essential nutrients including transition metals and metal-containing co-factors. Recently, an IgG antibody targeting the external binding protein of the Staphylococcus aureus Mn(II) ABC importer was reported to inhibit transport activity and reduce bacterial cell growth. We here explored the possibility of using alpaca-derived nanobodies to inhibit the vitamin B12 transporter of Escherichia coli, BtuCD-F, as a model system by generating nanobodies against the periplasmic binding protein BtuF. We isolated six nanobodies that competed with B12 for binding to BtuF, with inhibition constants between 10−6 and 10−9 M. Kinetic characterization of the nanobody-BtuF interactions revealed dissociation half-lives between 1.6 and 6 minutes and fast association rates between 104 and 106 M−1s−1. For the tightest-binding nanobody, we observed a reduction of in vitro transport activity of BtuCD-F when an excess of nanobody over B12 was used. The structure of BtuF in complex with the most effective nanobody Nb9 revealed the molecular basis of its inhibitory function. The CDR3 loop of Nb9 reached into the substrate-binding pocket of BtuF, preventing both B12 binding and BtuCD-F complex formation. Our results suggest that nanobodies can mediate ABC importer inhibition, providing an opportunity for novel antibiotic strategies

Structure of AMP-PNP-bound BtuCD and mechanism of ATP-powered vitamin B12 transport by BtuCD-F

ISSN:1545-9993ISSN:1545-998