Identifying bacterial and archaeal homologs of pentameric ligand-gated ion channel (pLGIC) family using domain-based and alignment-based approaches

Identification of bacterial and archaeal counterparts to eukaryotic ion channels has greatly facilitated studies of structural biophysics of the channels. Often, searches based only on sequence alignment tools are inadequate for discovering such distant bacterial and archaeal counterparts. We address the discovery of bacterial and archaeal members of the Pentameric Ligand-Gated Ion Channel (pLGIC) family by a combination of four computational methods. One domain-based method involves retrieval of proteins with pLGIC-relevant domains by matching those domains to previously established domain templates in the InterPro family of databases. The second domain-based method involves searches using ungapped de-novo motifs discovered by MEME which were trained with well characterized members of the pLGIC family. The third and fourth methods involve the use of two sequence alignment search algorithms BLASTp and psiBLAST respectively. The sequences returned from all methods were screened by having the correct topology for pLGIC's, and by returning an annotated member of this family as one of the first ten hits using BLASTp against a comprehensive database of eukaryotic proteins. We found the domain based searches to have high specificity but low sensitivity, while the sequence alignment methods have higher sensitivity but lower specificity. The four methods together discovered 69 putative bacterial and archaeal members of the pLGIC family. We ranked and divide the 69 proteins into groups according to the similarity of their domain compositions with known eukaryotic pLGIC's. One especially notable group is more closely related to eukaryotic pLGIC's than to any other known protein family, and has the overall topology of pLGIC's, but the functional domains they contain are sufficiently different from those found in known pLGIC's that they do not score very well against the pLGIC domain templates. We conclude that multiple methods used in a coordinated fashion outperform any single method for identifying likely distant bacterial and archaeal proteins that may provide useful models for important eukaryotic channel function. We note also that the methods used here are largely standard and readily accessible. The novelty is in the effectiveness of a strategy that combines these methods for identifying bacterial and archea relatives of this family. Therefore the paper may serve as a template for a broad group of workers to reliably identify bacterial and archaeal counterparts to eukaryotic proteins.     

Novel Predicted RNA-Binding Domains Associated with the Translation Machinery

Two previously undetected domains were identified in a variety of RNA-binding proteins, particularly RNA-modifying enzymes, using methods for sequence profile analysis. A small domain consisting of 60–65 amino acid residues was detected in the ribosomal protein S4, two families of pseudouridine synthases, a novel family of predicted RNA methylases, a yeast protein containing a pseudouridine synthetase and a deaminase domain, bacterial tyrosyl-tRNA synthetases, and a number of uncharacterized, small proteins that may be involved in translation regulation. Another novel domain, designated PUA domain, after PseudoUridine synthase and Archaeosine transglycosylase, was detected in archaeal and eukaryotic pseudouridine synthases, archaeal archaeosine synthases, a family of predicted ATPases that may be involved in RNA modification, a family of predicted archaeal and bacterial rRNA methylases. Additionally, the PUA domain was detected in a family of eukaryotic proteins that also contain a domain homologous to the translation initiation factor eIF1/SUI1; these proteins may comprise a novel type of translation factors. Unexpectedly, the PUA domain was detected also in bacterial and yeast glutamate kinases; this is compatible with the demonstrated role of these enzymes in the regulation of the expression of other genes. We propose that the S4 domain and the PUA domain bind RNA molecules with complex folded structures, adding to the growing collection of nucleic acid-binding domains associated with DNA and RNA modification enzymes. The evolution of the translation machinery components containing the S4, PUA, and SUI1 domains must have included several events of lateral gene transfer and gene loss as well as lineage-specific domain fusions. Received: 15 May 1998 / Accepted: 20 July 1998     

Domain-based identification and analysis of glutamate receptor ion channels and their relatives in prokaryotes

Voltage-gated and ligand-gated ion channels are used in eukaryotic organisms for the purpose of electrochemical signaling. There are prokaryotic homologues to major eukaryotic channels of these sorts, including voltage-gated sodium, potassium, and calcium channels, Ach-receptor and glutamate-receptor channels. The prokaryotic homologues have been less well characterized functionally than their eukaryotic counterparts. In this study we identify likely prokaryotic functional counterparts of eukaryotic glutamate receptor channels by comprehensive analysis of the prokaryotic sequences in the context of known functional domains present in the eukaryotic members of this family. In particular, we searched the nonredundant protein database for all proteins containing the following motif: the two sections of the extracellular glutamate binding domain flanking two transmembrane helices. We discovered 100 prokaryotic sequences containing this motif, with a wide variety of functional annotations. Two groups within this family have the same topology as eukaryotic glutamate receptor channels. Group 1 has a potassium-like selectivity filter. Group 2 is most closely related to eukaryotic glutamate receptor channels. We present analysis of the functional domain architecture for the group of 100, a putative phylogenetic tree, comparison of the protein phylogeny with the corresponding species phylogeny, consideration of the distribution of these proteins among classes of prokaryotes, and orthologous relationships between prokaryotic and human glutamate receptor channels. We introduce a construct called the Evolutionary Domain Network, which represents a putative pathway of domain rearrangements underlying the domain composition of present channels. We believe that scientists interested in ion channels in general, and ligand-gated ion channels in particular, will be interested in this work. The work should also be of interest to bioinformatics researchers who are interested in the use of functional domain-based analysis in evolutionary and functional discovery.     

Protein sequence similarity searches using patterns as seeds.

Protein families often are characterized by conserved sequence patterns or motifs. A researcher frequently wishes to evaluate the significance of a specific pattern within a protein, or to exploit knowledge of known motifs to aid the recognition of greatly diverged but homologous family members. To assist in these efforts, the pattern-hit initiated BLAST (PHI-BLAST) program described here takes as input both a protein sequence and a pattern of interest that it contains. PHI-BLAST searches a protein database for other instances of the input pattern, and uses those found as seeds for the construction of local alignments to the query sequence. The random distribution of PHI-BLAST alignment scores is studied analytically and empirically. In many instances, the program is able to detect statistically significant similarity between homologous proteins that are not recognizably related using traditional single-pass database search methods. PHI-BLAST is applied to the analysis of CED4-like cell death regulators, HS90-type ATPase domains, archaeal tRNA nucleotidyltransferases and archaeal homologs of DnaG-type DNA primases.     

Solution Structure and Calcium-Binding Properties of M-Crystallin, A Primordial βγ-Crystallin from Archaea

The lens βγ-crystallin superfamily has many diverse but topologically related members belonging to various taxa. Based on structural topology, these proteins are considered to be evolutionarily related to lens crystallins, suggesting their origin from a common ancestor. Proteins with βγ-crystallin domains, although found in some eukaryotes and eubacteria, have not yet been reported in archaea. Sequence searches in the genome of the archaebacterium Methanosarcina acetivorans revealed the presence of a protein annotated as a βγ-crystallin family protein, named M-crystallin. Solution structure of this protein indicates a typical βγ-crystallin fold with a paired Greek-key motif. Among the known structures of βγ-crystallin members, M-crystallin was found to be structurally similar to the vertebrate lens βγ-crystallins. The Ca^2 +-binding properties of this primordial protein are somewhat more similar to those of vertebrate βγ-crystallins than to those of bacterial homologues. These observations, taken together, suggest that amphibian and vertebrate βγ-crystallin domains are evolutionarily more related to archaeal homologues than to bacterial homologues. Additionally, identification of a βγ-crystallin homologue in archaea allows us to demonstrate the presence of this domain in all the three domains of life.     

ADEPTs: information necessary for subcellular distribution of eukaryotic sorting isozymes resides in domains missing from eubacterial and archaeal counterparts

Sorting isozymes are encoded by single genes, but the encoded proteins are distributed to multiple subcellular compartments. We surveyed the predicted protein sequences of several nucleic acid interacting sorting isozymes from the eukaryotic taxonomic domain and compared them with their homologs in the archaeal and eubacterial domains. Here, we summarize the data showing that the eukaryotic sorting isozymes often possess sequences not present in the archaeal and eubacterial counterparts and that the additional sequences can act to target the eukaryotic proteins to their appropriate subcellular locations. Therefore, we have named these protein domains ADEPTs (Additional Domains for Eukaryotic Protein Targeting). Identification of additional domains by phylogenetic comparisons should be generally useful for locating candidate sequences important for subcellular distribution of eukaryotic proteins.     

Evolution of the prokaryotic protein translocation complex: a comparison of archaeal and bacterial versions of SecDF

Protein translocation across the prokaryotic plasma membrane occurs at the translocon, an evolutionarily conserved membrane-embedded proteinaceous complex. Together with the core components SecYE, prokaryotic translocons also contain auxilliary proteins, such as SecDF. Alignment of bacterial and archaeal SecDF protein sequences reveals the presence of a similar number of homologous regions within each protein. Moreover, the conserved sequence domains in the archaeal proteins are located in similar positions as their bacterial counterparts. When these domains are, however, compared along Bacteria-Archaea lines, a much lower degree of similarity is detected. In Bacteria, SecDF are thought to modulate the membrane association of SecA, the ATPase that provides the driving force for bacterial protein secretion. As no archaeal version of SecA has been detected, the sequence differences reported here may reflect functional differences between bacterial and archaeal SecDF proteins, and by extension, between the bacterial and archaeal protein translocation processes. Moreover, the apparent absence of SecDF in several completed archaeal genomes suggests that differences may exist in the process of protein translocation within the archaeal domain itself.     

Archaea and the cell cycle

Sequence similarity data suggest that archaeal chromosome replication is eukaryotic in character. Putative nucleoid-processing proteins display similarities to both eukaryotic and bacterial counterparts, whereas cell division may occur through a predominantly bacterial mechanism. Insights into the organization of the archaeal cell cycle are therefore of interest, not only for understanding archaeal biology, but also for investigating how components from the other two domains interact and work in concert within the same cell; in addition, archaea may have the potential to provide insights into eukaryotic initiation of chromosome replication.     

DNA polymerase beta-like nucleotidyltransferase superfamily: identification of three new families, classification and evolutionary history

A detailed analysis of the polbeta superfamily of nucleotidyltransferases was performed using computer methods for iterative database search, multiple alignment, motif analysis and structural modeling. Three previously uncharacterized families of predicted nucleotidyltransferases are described. One of these new families includes small proteins found in all archaea and some bacteria that appear to consist of the minimal nucleotidyltransferase domain and may resemble the ancestral state of this superfamily. Another new family that is specifically related to eukaryotic polyA polymerases is typified by yeast Trf4p and Trf5p proteins that are involved in chromatin remodeling. The TRF family is represented by multiple members in all eukaryotes and may be involved in yet unknown nucleotide polymerization reactions required for maintenance of chromatin structure. Another new family of bacterial and archaeal nucleotidyltransferases is predicted to function in signal transduction since, in addition to the nucleotidyltransferase domain, these proteins contain ligand-binding domains. It is further shown that the catalytic domain of gamma proteobacterial adenylyl cyclases is homologous to the polbeta superfamily nucleotidyltransferases which emphasizes the general trend for the origin of signal-transducing enzymes from those involved in replication, repair and RNA processing. Classification of the polbeta superfamily into distinct families and examination of their phyletic distribution suggests that the evolution of this type of nucleotidyltransferases may have included bursts of rapid divergence linked to the emergence of new functions as well as a number of horizontal gene transfer events.     

The ubiquitin domain superfold: structure-based sequence alignments and characterization of binding epitopes

Ubiquitin-like domains are present, apart from ubiquitin-like proteins themselves, in many multidomain proteins involved in different signal transduction processes. The sequence conservation for all ubiquitin superfold family members is rather poor, even between subfamily members, leading to mistakes in sequence alignments using conventional sequence alignment methods. However, a correct alignment is essential, especially for in silico methods that predict binding partners on the basis of sequence and structure. In this study, using 3D-structural information we have generated and manually corrected sequence alignments for proteins of the five ubiquitin superfold subfamilies. On the basis of this alignment, we suggest domains for which structural information will be useful to allow homology modelling. In addition, we have analysed the energetic and electrostatic properties of ubiquitin-like domains in complex with various functional binding proteins using the protein design algorithm FoldX. On the basis of an in silico alanine-scanning mutagenesis, we provide a detailed binding epitope mapping of the hotspots of the ubiquitin domain fold, involved in the interaction with different domains and proteins. Finally, we provide a consensus fingerprint sequence that identifies all sequences described to belong to the ubiquitin superfold family. It is possible that the method that we describe may be applied to other domain families sharing a similar fold but having low levels of sequence homology.     

Regions of minimal structural variation among members of protein domain superfamilies: application to remote homology detection and modelling using distant relationships

Structurally conserved regions or structural templates have been identified and examined for features such as amino acid content, solvent accessibility, secondary structures, non-polar interaction, residue packing and extent of structural deviations in 179 aligned members of superfamilies involving 1208 pairs of protein domains. An analysis of these structural features shows that the retention of secondary structural conservation and similar hydrogen bonding pattern within the templates is 2.5 and 1.8 times higher, respectively, than full-length alignments suggesting that they form the minimum structural requirement of a superfamily. The identification and availability of structural templates find value in different areas of protein structure prediction and modelling such as in sensitive sequence searches, accurate sequence alignment and three-dimensional modelling on the basis of distant relationships.     

Of proteins and RNA: The RNase P/MRP family

Nuclear ribonuclease (RNase) P is a ubiquitous essential ribonucleoprotein complex, one of only two known RNA-based enzymes found in all three domains of life. The RNA component is the catalytic moiety of RNases P across all phylogenetic domains; it contains a well-conserved core, whereas peripheral structural elements are diverse. RNA components of eukaryotic RNases P tend to be less complex than their bacterial counterparts, a simplification that is accompanied by a dramatic reduction of their catalytic ability in the absence of protein. The size and complexity of the protein moieties increase dramatically from bacterial to archaeal to eukaryotic enzymes, apparently reflecting the delegation of some structural functions from RNA to proteins and, perhaps, in response to the increased complexity of the cellular environment in the more evolutionarily advanced organisms; the reasons for the increased dependence on proteins are not clear. We review current information on RNase P and the closely related universal eukaryotic enzyme RNase MRP, focusing on their functions and structural organization.     

The discoidin domain family revisited: new members from prokaryotes and a homology-based fold prediction.

Members of the discoidin (DS) domain family, which includes the C1 and C2 repeats of blood coagulation factors V and VIII, occur in a great variety of eukaryotic proteins, most of which have been implicated in cell-adhesion or developmental processes. So far, no three-dimensional structure of a known example of this extracellular module has been determined, limiting the usefulness of identifying a new sequence as member of this family. Here, we present results of a recent search of the protein sequence database for new DS domains using generalized profiles, a sensitive multiple alignment-based search technique. Several previously unrecognized DS domains could be identified by this method, including the first examples from prokaryotic species. More importantly, we present statistical, structural, and functional evidence that the D1 domain of galactose oxidase whose three-dimensional structure has been determined at 1.7 A resolution, is a distant member of this family. Taken together, these findings significantly expand the concept of the DS domain, by extending its taxonomic range and by implying a fold prediction for all its members. The proposed alignment with the galactose oxidase sequence makes it possible to construct homology-based three-dimensional models for the most interesting examples, as illustrated by an accompanying paper on the C1 and C2 domains of factor V.     

Structural Features of the Glutamate Transporter Family

Neuronal and glial glutamate transporters remove the excitatory neurotransmitter glutamate from the synaptic cleft and thus prevent neurotoxicity. The proteins belong to a large and widespread family of secondary transporters, including bacterial glutamate, serine, and C₄-dicarboxylate transporters; mammalian neutral-amino-acid transporters; and an increasing number of bacterial, archaeal, and eukaryotic proteins that have not yet been functionally characterized. Sixty members of the glutamate transporter family were found in the databases on the basis of sequence homology. The amino acid sequences of the carriers have diverged enormously. Homology between the members of the family is most apparent in a stretch of approximately 150 residues in the C-terminal part of the proteins. This region contains four reasonably well-conserved sequence motifs, all of which have been suggested to be part of the translocation pore or substrate binding site. Phylogenetic analysis of the C-terminal stretch revealed the presence of five subfamilies with characterized members: (i) the eukaryotic glutamate transporters, (ii) the bacterial glutamate transporters, (iii) the eukaryotic neutral-amino-acid transporters, (iv) the bacterial C₄-dicarboxylate transporters, and (v) the bacterial serine transporters. A number of other subfamilies that do not contain characterized members have been defined. In contrast to their amino acid sequences, the hydropathy profiles of the members of the family are extremely well conserved. Analysis of the hydropathy profiles has suggested that the glutamate transporters have a global structure that is unique among secondary transporters. Experimentally, the unique structure of the transporters was recently confirmed by membrane topology studies. Although there is still controversy about part of the topology, the most likely model predicts the presence of eight membrane-spanning α-helices and a loop-pore structure which is unique among secondary transporters but may resemble loop-pores found in ion channels. A second distinctive structural feature is the presence of a highly amphipathic membrane-spanning helix that provides a hydrophilic path through the membrane. Recent data from analysis of site-directed mutants and studies on the mechanism and pharmacology of the transporters are discussed in relation to the structural model.     

Structural features of the glutamate transporter family.

Neuronal and glial glutamate transporters remove the excitatory neurotransmitter glutamate from the synaptic cleft and thus prevent neurotoxicity. The proteins belong to a large and widespread family of secondary transporters, including bacterial glutamate, serine, and C4-dicarboxylate transporters; mammalian neutral-amino-acid transporters; and an increasing number of bacterial, archaeal, and eukaryotic proteins that have not yet been functionally characterized. Sixty members of the glutamate transporter family were found in the databases on the basis of sequence homology. The amino acid sequences of the carriers have diverged enormously. Homology between the members of the family is most apparent in a stretch of approximately 150 residues in the C-terminal part of the proteins. This region contains four reasonably well-conserved sequence motifs, all of which have been suggested to be part of the translocation pore or substrate binding site. Phylogenetic analysis of the C-terminal stretch revealed the presence of five subfamilies with characterized members: (i) the eukaryotic glutamate transporters, (ii) the bacterial glutamate transporters, (iii) the eukaryotic neutral-amino-acid transporters, (iv) the bacterial C4-dicarboxylate transporters, and (v) the bacterial serine transporters. A number of other subfamilies that do not contain characterized members have been defined. In contrast to their amino acid sequences, the hydropathy profiles of the members of the family are extremely well conserved. Analysis of the hydropathy profiles has suggested that the glutamate transporters have a global structure that is unique among secondary transporters. Experimentally, the unique structure of the transporters was recently confirmed by membrane topology studies. Although there is still controversy about part of the topology, the most likely model predicts the presence of eight membrane-spanning alpha-helices and a loop-pore structure which is unique among secondary transporters but may resemble loop-pores found in ion channels. A second distinctive structural feature is the presence of a highly amphipathic membrane-spanning helix that provides a hydrophilic path through the membrane. Recent data from analysis of site-directed mutants and studies on the mechanism and pharmacology of the transporters are discussed in relation to the structural model.     

Structural and Functional Divergence within the Dim1/KsgA Family of rRNA Methyltransferases

The enzymes of the KsgA/Dim1 family are universally distributed throughout all phylogeny; however, structural and functional differences are known to exist. The well-characterized function of these enzymes is to dimethylate two adjacent adenosines of the small ribosomal subunit in the normal course of ribosome maturation, and the structures of KsgA from Escherichia coli and Dim1 from Homo sapiens and Plasmodium falciparum have been determined. To this point, no examples of archaeal structures have been reported. Here, we report the structure of Dim1 from the thermophilic archaeon Methanocaldococcus jannaschii. While it shares obvious similarities with the bacterial and eukaryotic orthologs, notable structural differences exist among the three members, particularly in the C-terminal domain. Previous work showed that eukaryotic and archaeal Dim1 were able to robustly complement for KsgA in E. coli. Here, we repeated similar experiments to test for complementarity of archaeal Dim1 and bacterial KsgA in Saccharomyces cerevisiae. However, neither the bacterial nor the archaeal ortholog could complement for the eukaryotic Dim1. This might be related to the secondary, non-methyltransferase function that Dim1 is known to play in eukaryotic ribosomal maturation. To further delineate regions of the eukaryotic Dim1 critical to its function, we created and tested KsgA/Dim1 chimeras. Of the chimeras, only one constructed with the N-terminal domain from eukaryotic Dim1 and the C-terminal domain from archaeal Dim1 was able to complement, suggesting that eukaryotic-specific Dim1 function resides in the N-terminal domain also, where few structural differences are observed between members of the KsgA/Dim1 family. Future work is required to identify those determinants directly responsible for Dim1 function in ribosome biogenesis. Finally, we have conclusively established that none of the methyl groups are critically important to growth in yeast under standard conditions at a variety of temperatures.     

Novel Families of Archaeo-Eukaryotic Primases Associated with Mobile Genetic Elements of Bacteria and Archaea

Cellular organisms in different domains of life employ structurally unrelated, non-homologous DNA primases for synthesis of a primer for DNA replication. Archaea and eukaryotes encode enzymes of the archaeo-eukaryotic primase (AEP) superfamily, whereas bacteria uniformly use primases of the DnaG family. However, AEP genes are widespread in bacterial genomes raising questions regarding their provenance and function. Here, using an archaeal primase–polymerase PolpTN2 encoded by pTN2 plasmid as a seed for sequence similarity searches, we recovered over 800 AEP homologs from bacteria belonging to 12 highly diverse phyla. These sequences formed a supergroup, PrimPol-PV1, and could be classified into five novel AEP families which are characterized by a conserved motif containing an arginine residue likely to be involved in nucleotide binding. Functional assays confirm the essentiality of this motif for catalytic activity of the PolpTN2 primase–polymerase. Further analyses showed that bacterial AEPs display a range of domain organizations and uncovered several candidates for novel families of helicases. Furthermore, sequence and structure comparisons suggest that PriCT-1 and PriCT-2 domains frequently fused to the AEP domains are related to each other as well as to the non-catalytic, large subunit of archaeal and eukaryotic primases, and to the recently discovered PriX subunit of archaeal primases. Finally, genomic neighborhood analysis indicates that the identified AEPs encoded in bacterial genomes are nearly exclusively associated with highly diverse integrated mobile genetic elements, including integrative conjugative plasmids and prophages.