The iron/lead transporter superfamily of Fe/Pb2+ uptake systems

Oxidase-dependent ferrous iron uptake transporters of the OFeT family and lead uptake transporters of the PbrT family comprise the iron/lead transporter (ILT) superfamily (transporter classification No. 9.A.10). All sequenced homologues of the ILT superfamily were multiply aligned, and conserved motifs, including fully conserved acidic residues in putative transmembrane segments (TMSs) 1 and 4, previously implicated in heavy metal binding, were identified. Topological analyses confirmed the presence of 7 conserved TMSs in a 3 + 3 + 1 arrangement where the two 3 TMS elements are internally repeated. Phylogenetic analyses revealed the presence of several sequence divergent clusters of orthologous proteins that group roughly according to the phylogenes of the organisms of origin. The results serve to characterize and provide evolutionary insight into a novel superfamily of heavy metal uptake transporters.     

Construction and analysis of a profile library characterizing groups of structurally known proteins.

A new sequence motif library StrProf was constructed characterizing the groups of related proteins in the PDB three-dimensional structure database. For a representative member of each protein family, which was identified by cross-referencing the PDB with the PIR superfamily classification, a group of related sequences was collected by the BLAST search against the nonredundant protein sequence database. For every group, the motifs were identified automatically according to the criteria of conservation and uniqueness of pentapeptide patterns and with a dual dynamic programming algorithm. In the StrProf library, motifs are represented by profile matrices rather than consensus patterns to allow more flexible search capabilities. Another dynamic programming algorithm was then developed to search this motif library. When the computationally derived StrProf was compared with PROSITE, which is a manually derived motif library in the best consensus pattern representation, the numbers of identified patterns were comparable. StrProf missed about one third of the PROSITE motifs, but there were also new motifs lacking in PROSITE. The new library was incorporated in SMART (Sequence Motif Analysis and Retrieval Tool), a computer tool designed to help search and annotate biologically important sites in an unknown protein sequence. The client program is available free of charge through the Internet.     

Plant ABC transporters

The ATP binding cassette (ABC) superfamily is a large, ubiquitous and diverse group of proteins, most of which mediate transport across biological membranes. ABC transporters have been shown to function not only as ATP-dependent pumps, but also as ion channels and channel regulators. Whilst members of this gene family have been extensively characterised in mammalian and microbial systems, the study of plant ABC transporters is a relatively new field of investigation. Sequences of over 20 plant ABC proteins have been published and include homologues of P-glycoprotein, MRP, PDR5 and organellar transporters. At present, functions have been assigned to a small proportion of these genes and only the MRP subclass has been extensively characterised. This review aims to summarise literature relevant to the study of plant ABC transporters, to review methods of cloning, to discuss the utility of yeast and mammalian systems as models and to speculate on possible roles of uncharacterised ABC transporters in plants.     

Inventory and Comparative Evolution of the ABC Superfamily in the Genomes of <Emphasis Type="Italic">Phytophthora ramorum</Emphasis> and <Emphasis Type="Italic">Phytophthora sojae</Emphasis>

Automated and manual annotation of the ATP binding cassette (ABC) superfamily in the Phytophthora ramorum and P. sojae genomes has identified 135 and 136 members, respectively, indicating that this family is comparable in size to the Arabidopsis thaliana and rice genomes, and significantly larger than that of two fungal pathogens, Fusarium graminearum and Magnaporthe grisea. The high level of synteny between these oomycete genomes extends to the ABC superfamily, where 108 orthologues were identified by phylogenetic analysis. The largest subfamilies include those most often associated with multidrug resistance. The P. ramorum genome contains 22 multidrug resistance-associated protein (MRP) genes and 49 pleiotropic drug resistance (PDR) genes, while P. sojae contains 20 MRP and 49 PDR genes. Tandem duplication events in the last common ancestor appear to account for much of the expansion of these subfamilies. Recent duplication events in the PDR and ABCG families in both the P. ramorum and the P. sojae genomes indicate that selective expansion of ABC transporters may still be occurring. In other kingdoms, subfamilies define both domain arrangements and proteins having a common phylogenetic origin, but this is not the case for several subfamilies in oomycetes. At least one ABCG type transporter is derived from a PDR transporter, while transporters in the ABCB-half family cluster with transporters from bacterial, plant, and metazoan genomes. Additional examples of transporters that appear to be derived from horizontal transfer events from bacterial genomes include components of transporters associated with iron uptake and DNA repair. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Evolution of the Oligopeptide Transporter Family

The oligopeptide transporter (OPT) family of peptide and iron-siderophore transporters includes members from both prokaryotes and eukaryotes but with restricted distribution in the latter domain. Eukaryotic members were found only in fungi and plants with a single slime mold homologue clustering with the fungal proteins. All functionally characterized eukaryotic peptide transporters segregate from the known iron-siderophore transporters on a phylogenetic tree. Prokaryotic members are widespread, deriving from many different phyla. Although they belong only to the iron-siderophore subdivision, genome context analyses suggest that many of them are peptide transporters. OPT family proteins have 16 or occasionally 17 transmembrane-spanning α-helical segments (TMSs). We provide statistical evidence that the 16-TMS topology arose via three sequential duplication events followed by a gene-fusion event for proteins with a seventeenth TMS. The proposed pathway is as follows: 2 TMSs → 4 TMSs → 8 TMSs → 16 TMSs → 17 TMSs. The seventeenth C-terminal TMS, which probably arose just once, is found in just one phylogenetic group of these homologues. Analyses for orthology revealed that a few phylogenetic clusters consist exclusively of orthologues but most have undergone intermixing, suggestive of horizontal transfer. It appears that in this family horizontal gene transfer was frequent among prokaryotes, rare among eukaryotes and largely absent between prokaryotes and eukaryotes as well as between plants and fungi. These observations provide guides for future structural and functional analyses of OPT family members.     

Expanding the nitrogen regulatory protein superfamily: Homology detection at below random sequence identity

Nitrogen regulatory (PII) proteins are signal transduction molecules involved in controlling nitrogen metabolism in prokaryots. PII proteins integrate the signals of intracellular nitrogen and carbon status into the control of enzymes involved in nitrogen assimilation. Using elaborate sequence similarity detection schemes, we show that five clusters of orthologs (COGs) and several small divergent protein groups belong to the PII superfamily and predict their structure to be a (betaalphabeta)(2) ferredoxin-like fold. Proteins from the newly emerged PII superfamily are present in all major phylogenetic lineages. The PII homologs are quite diverse, with below random (as low as 1%) pairwise sequence identities between some members of distant groups. Despite this sequence diversity, evidence suggests that the different subfamilies retain the PII trimeric structure important for ligand-binding site formation and maintain a conservation of conservations at residue positions important for PII function. Because most of the orthologous groups within the PII superfamily are composed entirely of hypothetical proteins, our remote homology-based structure prediction provides the only information about them. Analogous to structural genomics efforts, such prediction gives clues to the biological roles of these proteins and allows us to hypothesize about locations of functional sites on model structures or rationalize about available experimental information. For instance, conserved residues in one of the families map in close proximity to each other on PII structure, allowing for a possible metal-binding site in the proteins coded by the locus known to affect sensitivity to divalent metal ions. Presented analysis pushes the limits of sequence similarity searches and exemplifies one of the extreme cases of reliable sequence-based structure prediction. In conjunction with structural genomics efforts to shed light on protein function, our strategies make it possible to detect homology between highly diverse sequences and are aimed at understanding the most remote evolutionary connections in the protein world.     

Sugar transporters from bacteria, parasites and mammals: structure–activity relationships

Sugar transport across the plasma membrane is one of the most important transport processes. The cloning and expression of cDNAs from a superfamily of related sugar transporters that all adopt a 12-membrane-spanning-domain structure has opened new avenues of investigation, including pre-steady-state kinetic analysis. Structure–function analyses of mammalian and bacterial sugar transporters, and comparisons of these transporters with those of parasitic trypanosomatids, indicate that different environmental pressures have tailored the evolution of the various members of the sugar-transporter superfamily. Subtle distinctions in the function of these proteins can be related to particular amino acid residue substitutions.     

Complete inventory of ABC proteins in human pathogenic yeast, Candida albicans

The recent completion of the sequencing project of the opportunistic human pathogenic yeast, Candida albicans (http://www.ncbi.nlm.nih.gov/), led us to analyze and classify its ATP-binding cassette (ABC) proteins, which constitute one of the largest superfamilies of proteins. Some of its members are multidrug transporters responsible for the commonly encountered problem of antifungal resistance. TBLASTN searches together with domain analysis identified 81 nucleotide-binding domains, which belong to 51 different putative open reading frames. Considering that each allelic pair represents a single ABC protein of the Candida genome, the total number of putative members of this superfamily is 28. Domain organization, sequence-based analysis and self-organizing map-based clustering led to the classification of Candida ABC proteins into 6 distinct subfamilies. Each subfamily from C. albicans has an equivalent in Saccharomyces cerevisiae suggesting a close evolutionary relationship between the two yeasts. Our searches also led to the identification of a new motif to each subfamily in Candida that could be used to identify sequences from the corresponding subfamily in other organisms. It is hoped that the inventory of Candida ABC transporters thus created will provide new insights into the role of ABC proteins in antifungal resistance as well as help in the functional characterization of the superfamily of these proteins.     

A method for probabilistic mapping between protein structure and function taxonomies through cross training

Background

Prediction of function of proteins on the basis of structure and vice versa is a partially solved problem, largely in the domain of biophysics and biochemistry. This underlies the need of computational and bioinformatics approach to solve the problem. Large and organized latent knowledge on protein classification exists in the form of independently created protein classification databases. By creating probabilistic maps between classes of structural classification databases (e.g. SCOP [1]) and classes of functional classification databases (e.g. PROSITE [2]), structure and function of proteins could be probabilistically related.

Results

We demonstrate that PROSITE and SCOP have significant semantic overlap, in spite of independent classification schemes. By training classifiers of SCOP using classes of PROSITE as attributes and vice versa, accuracy of Support Vector Machine classifiers for both SCOP and PROSITE was improved. Novel attributes, 2-D elastic profiles and Blocks were used to improve time complexity and accuracy. Many relationships were extracted between classes of SCOP and PROSITE using decision trees.

Conclusion

We demonstrate that presented approach can discover new probabilistic relationships between classes of different taxonomies and render a more accurate classification. Extensive mappings between existing protein classification databases can be created to link the large amount of organized data. Probabilistic maps were created between classes of SCOP and PROSITE allowing predictions of structure using function, and vice versa. In our experiments, we also found that functions are indeed more strongly related to structure than are structure to functions.     

Genetic transformation of genes for protein II in Neisseria gonorrhoeae.

The protein II (PII) outer membrane proteins of Neisseria gonorrhoeae are a family of heat-modifiable proteins that are subject to phase variation, in which the synthesis of different PII species is turned on and off at a high frequency. Transformation of PII genes from a donor gonococcal strain into a recipient strain was detected with monoclonal antibodies specific for the PII proteins of the donor. Individual PII protein-expressing transformants generally bound only one donor-specific PII monoclonal antibody. Recovery of transformants expressing a donor-specific PII protein depended on the PII protein expression state of the donor: the transformed population bound only monoclonal antibodies specific for PII proteins that were expressed in the donor. Colony variants with an altered frequency of switching of PII protein expression were isolated, but the altered switch phenotype did not cotransform with the PII structural gene. These results provide genetic evidence that PII proteins are the products of different genes and that expressed and unexpressed forms of the PII gene are different from each other.     

In vitro proof of direct regulation of glutamine synthetase by GlnK proteins in the extreme halophilic archaeon Haloferax mediterranei

Haloferax mediterranei is an extreme halophilic micro-organism belonging to the Archaea domain that was isolated from the Santa Pola solar salterns (Alicante, Spain) in 1983. The biochemistry of the proteins involved in nitrogen metabolism is being studied, but the knowledge of their regulation is very scarce at present. The PII superfamily is constituted by major regulators of nitrogen metabolism, which are widespread in prokaryotic and eukaryotic organisms. These trimeric proteins (12?kDa per subunit) have in Escherichia coli long been known to regulate GS (glutamine synthetase) activity via its adenylyltransferase/adenylyl-removing enzyme and, more recently, to be able to interact directly with this enzyme in methanogenic archaea. We have tested the possible role of PII proteins in the regulation of ammonium assimilation in our model organism and the results clearly indicate that the direct influence of GS by PII proteins can also take place in halophilic archaea, starting with the comprehension of nitrogen regulation in those organisms.     

New Insights into Evolution of the ABC Transporter Family in Mesostigma viride,a Unicellular Charophyte Algae

ATP-binding cassette (ABC) transporters play an important role in driving the exchange of multiple molecules across cell membranes. The plant ABC transporter family is among the largest protein families, and recent progress has advanced our understanding of ABC classification. However, the ancestral form and deep origin of plant ABCs remain elusive. In this study, we identified 59 ABC transporters in Mesostigma viride, a unicellular charophyte algae that represents the earliest diverging lineage of streptophytes, and 1034 ABCs in genomes representing a broad taxonomic sampling from distantly related plant evolutionary lineages, including chlorophytes, charophytes, bryophytes, lycophytes, gymnosperms, basal angiosperms, monocots, and eudicots. We classified the plant ABC transporters by comprehensive phylogenetic analysis of each subfamily. Our analysis revealed the ancestral type of ABC proteins as well as duplication and gene loss during plant evolution, contributing to our understanding of the functional conservation and diversity of this family. In summary, this study provides new insight into the origin and evolution of plant ABC transporters.     

Extra domains in secondary transport carriers and channel proteins

“Extra” domains in members of the families of secondary transport carrier and channel proteins provide secondary functions that expand, amplify or restrict the functional nature of these proteins. Domains in secondary carriers include TrkA and SPX domains in DASS family members, DedA domains in TRAP-T family members (both of the IT superfamily), Kazal-2 and PDZ domains in OAT family members (of the MF superfamily), USP, IIA^Fru and TrkA domains in ABT family members (of the APC superfamily), ricin domains in OST family members, and TrkA domains in AAE family members. Some transporters contain highly hydrophilic domains consisting of multiple repeat units that can also be found in proteins of dissimilar function. Similarly, transmembrane α-helical channel-forming proteins contain unique, conserved, hydrophilic domains, most of which are not found in carriers. In some cases the functions of these domains are known. They may be ligand binding domains, phosphorylation domains, signal transduction domains, protein/protein interaction domains or complex carbohydrate-binding domains. These domains mediate regulation, subunit interactions, or subcellular targeting. Phylogenetic analyses show that while some of these domains are restricted to closely related proteins derived from specific organismal types, others are nearly ubiquitous within a particular family of transporters and occur in a tremendous diversity of organisms. The former probably became associated with the transporters late in the evolutionary process; the latter probably became associated with the carriers much earlier. These domains can be located at either end of the transporter or in a central region, depending on the domain and transporter family. These studies provide useful information about the evolution of extra domains in channels and secondary carriers and provide novel clues concerning function.     

ProtoMap: automatic classification of protein sequences, a hierarchy of protein families, and local maps of the protein space

We investigate the space of all protein sequences in search of clusters of related proteins. Our aim is to automatically detect these sets, and thus obtain a classification of all protein sequences. Our analysis, which uses standard measures of sequence similarity as applied to an all-vs.-all comparison of SWISSPROT, gives a very conservative initial classification based on the highest scoring pairs. The many classes in this classification correspond to protein subfamilies. Subsequently we merge the subclasses using the weaker pairs in a two-phase clustering algorithm. The algorithm makes use of transitivity to identify homologous proteins; however, transitivity is applied restrictively in an attempt to prevent unrelated proteins from clustering together. This process is repeated at varying levels of statistical significance. Consequently, a hierarchical organization of all proteins is obtained. The resulting classification splits the protein space into well-defined groups of proteins, which are closely correlated with natural biological families and superfamilies. Different indices of validity were applied to assess the quality of our classification and compare it with the protein families in the PROSITE and Pfam databases. Our classification agrees with these domain-based classifications for between 64.8% and 88.5% of the proteins. It also finds many new clusters of protein sequences which were not classified by these databases. The hierarchical organization suggested by our analysis reveals finer subfamilies in families of known proteins as well as many novel relations between protein families.     

Neurotransmitter transporters in schistosomes: Structure,function and prospects for drug discovery

Neurotransmitter transporters (NTTs) play a fundamental role in the control of neurotransmitter signaling and homeostasis. Sodium symporters of the plasma membrane mediate the cellular uptake of neurotransmitter from the synaptic cleft, whereas proton-driven vesicular transporters sequester the neurotransmitter into synaptic vesicles for subsequent release. Together these transporters control how much transmitter is released and how long it remains in the synaptic cleft, thereby regulating the intensity and duration of signaling. NTTs have been the subject of much research in mammals and there is growing interest in their activities among invertebrates as well. In this review we will focus our attention on NTTs of the parasitic flatworm Schistosoma mansoni. Bloodflukes of the genus Schistosoma are the causative agents of human schistosomiasis, a devastating disease that afflicts over 200 million people worldwide. Schistosomes have a well-developed nervous system and a rich diversity of neurotransmitters, including many of the small-molecule (“classical”) neurotransmitters that normally employ NTTs in their mechanism of signaling. Recent advances in schistosome genomics have unveiled numerous NTTs in this parasite, some of which have now been cloned and characterized in vitro. Moreover new genetic and pharmacological evidence suggests that NTTs are required for proper control of neuromuscular signaling and movement of the worm. Among these carriers are proteins that have been successfully targeted for drug discovery in other organisms, in particular sodium symporters for biogenic amine neurotransmitters such as serotonin and dopamine. Our goal in this chapter is to review the current status of research on schistosome NTTs, with emphasis on biogenic amine sodium symporters, and to evaluate their potential for anti-schistosomal drug targeting. Through this discussion we hope to draw attention to this important superfamily of parasite proteins and to identify new directions for future research.     

Lactococcus lactis as host for overproduction of functional membrane proteins

Lactococcus lactis has many properties that are ideal for enhanced expression of membrane proteins. The organism is easy and inexpensive to culture, has a single membrane and relatively mild proteolytic activity. Methods for genetic manipulation are fully established and a tightly controlled promoter system is available, with which the level of expression can be varied with the inducer concentration. Here we describe our experiences with lactococcal expression of the mechanosensitive channel, the human KDEL receptor and transporters belonging to the ABC transporter family, the major facilitator superfamily, the mitochondrial carrier family and the peptide transporter family. Previously published expression studies only deal with the overexpression of prokaryotic membrane proteins, but in this paper, experimental data are presented for the overproduction of mitochondrial and hydrogenosomal carriers and the human KDEL receptor. These eukaryotic membrane proteins were expressed in a functional form and at levels amenable to structural work.