Characterization of the Tetraspan Junctional Complex (4JC) superfamily

Connexins or innexins form gap junctions, while claudins and occludins form tight junctions. In this study, statistical data, derived using novel software, indicate that these four junctional protein families and eleven other families of channel and channel auxiliary proteins are related by common descent and comprise the Tetraspan (4 TMS) Junctional Complex (4JC) Superfamily. These proteins all share similar 4 transmembrane α-helical (TMS) topologies. Evidence is presented that they arose via an intragenic duplication event, whereby a 2 TMS-encoding genetic element duplicated tandemly to give 4 TMS proteins. In cases where high resolution structural data were available, the conclusion of homology was supported by conducting structural comparisons. Phylogenetic trees reveal the probable relationships of these 15 families to each other. Long homologues containing fusions to other recognizable domains as well as internally duplicated or fused domains are reported. Large “fusion” proteins containing 4JC domains proved to fall predominantly into family-specific patterns as follows: (1) the 4JC domain was N-terminal; (2) the 4JC domain was C-terminal; (3) the 4JC domain was duplicated or occasionally triplicated and (4) mixed fusion types were present. Our observations provide insight into the evolutionary origins and subfunctions of these proteins as well as guides concerning their structural and functional relationships

Expansion of the Transporter-Opsin-G protein-coupled receptor superfamily with five new protein families.

Here we provide bioinformatic evidence that the Organo-Arsenical Exporter (ArsP), Endoplasmic Reticulum Retention Receptor (KDELR), Mitochondrial Pyruvate Carrier (MPC), L-Alanine Exporter (AlaE), and the Lipid-linked Sugar Translocase (LST) protein families are members of the Transporter-Opsin-G Protein-coupled Receptor (TOG) Superfamily. These families share domains homologous to well-established TOG superfamily members, and their topologies of transmembranal segments (TMSs) are compatible with the basic 4-TMS repeat unit characteristic of this Superfamily. These repeat units tend to occur twice in proteins as a result of intragenic duplication events, often with subsequent gain/loss of TMSs in many superfamily members. Transporters within the ArsP family allow microbial pathogens to expel toxic arsenic compounds from the cell. Members of the KDELR family are involved in the selective retrieval of proteins that reside in the endoplasmic reticulum. Proteins of the MPC family are involved in the transport of pyruvate into mitochondria, providing the organelle with a major oxidative fuel. Members of family AlaE excrete L-alanine from the cell. Members of the LST family are involved in the translocation of lipid-linked glucose across the membrane. These five families substantially expand the range of substrates of transport carriers in the superfamily, although KDEL receptors have no known transport function. Clustering of protein sequences reveals the relationships among families, and the resulting tree correlates well with the degrees of sequence similarity documented between families. The analyses and programs developed to detect distant relatedness, provide insights into the structural, functional, and evolutionary relationships that exist between families of the TOG superfamily, and should be of value to many other investigators

Bioinformatic characterization of the Anoctamin Superfamily of Ca2+-activated ion channels and lipid scramblases.

Our laboratory has developed bioinformatic strategies for identifying distant phylogenetic relationships and characterizing families and superfamilies of transport proteins. Results using these tools suggest that the Anoctamin Superfamily of cation and anion channels, as well as lipid scramblases, includes three functionally characterized families: the Anoctamin (ANO), Transmembrane Channel (TMC) and Ca2+-permeable Stress-gated Cation Channel (CSC) families; as well as four families of functionally uncharacterized proteins, which we refer to as the Anoctamin-like (ANO-L), Transmembrane Channel-like (TMC-L), and CSC-like (CSC-L1 and CSC-L2) families. We have constructed protein clusters and trees showing the relative relationships among the seven families. Topological analyses suggest that the members of these families have essentially the same topologies. Comparative examination of these homologous families provides insight into possible mechanisms of action, indicates the currently recognized organismal distributions of these proteins, and suggests drug design potential for the disease-related channel proteins

Characterization of the Tetraspan Junctional Complex (4JC) superfamily.

Connexins or innexins form gap junctions, while claudins and occludins form tight junctions. In this study, statistical data, derived using novel software, indicate that these four junctional protein families and eleven other families of channel and channel auxiliary proteins are related by common descent and comprise the Tetraspan (4 TMS) Junctional Complex (4JC) Superfamily. These proteins all share similar 4 transmembrane α-helical (TMS) topologies. Evidence is presented that they arose via an intragenic duplication event, whereby a 2 TMS-encoding genetic element duplicated tandemly to give 4 TMS proteins. In cases where high resolution structural data were available, the conclusion of homology was supported by conducting structural comparisons. Phylogenetic trees reveal the probable relationships of these 15 families to each other. Long homologues containing fusions to other recognizable domains as well as internally duplicated or fused domains are reported. Large "fusion" proteins containing 4JC domains proved to fall predominantly into family-specific patterns as follows: (1) the 4JC domain was N-terminal; (2) the 4JC domain was C-terminal; (3) the 4JC domain was duplicated or occasionally triplicated and (4) mixed fusion types were present. Our observations provide insight into the evolutionary origins and subfunctions of these proteins as well as guides concerning their structural and functional relationships

MAST output containing the top 3 motifs identified by MEME.

<p>The figure shows sequences with motif E-values < 10⁻³⁹. Motif 1 (red boxes) maps to TMSs 7 and 8, where 4 of the 6 Ca²⁺-binding residues in nhTMEM16 are located. Motif 2 (cyan boxes) maps to TMSs 4 and 5 in nhTMEM16, which form part of the subunit cavity for phospholipid translocation. Motif 3 (green boxes) maps to TMS 1, but this TMS does not interact with Ca²⁺ or the substrate. Our results show that 94% (65/69) of the sequences in the superfamily map Motif 1 to the region that contains 4 of the 6 functional residues that bind Ca²⁺, and 98.5% (68/69) of the sequences map Motif 2 to TMSs 4 and 5.</p

Piezo_Suppl_File_S2

This tarball files contains folders with the results of running MEME and MAST in three sets of proteins (i.e., the Training set, Positive control, and the Negative control). The data are organized in three directories (See Methods in the manuscript for more details):   MEME:  Contains the results of running MEME on a sample of 50 sequences for each one of the nine repeats characteristic of the Piezo family (TC: 1.A.75), resulting in 450 sequences total. Three Motifs 25 aa long where identified showing an E-value   MAST:  This folder contains two subfolders:   1. Folder Piezo_homologs contains the results of running MAST on 1229 non-redundant homologs of the Piezo Family using the motif models in folder MEME (E-value   2. Folder Negative_control contains the results of running MAST on the entire protein content of TCDB (22,215 proteins) using the motif models in folder MEME (E-value   Sequences This folder contains three sequence files used in the analysis.   1. File Piezo_50seqs_per_repeat.faa contains the random samples of 50 sequences per repeat (450 sequences total) used to run MEME. The Repeat to which each sequence belongs is identified with the prefix Rn_, where n = 1...9   2. File negative_control.faa contains all 22,215 TCDB sequences in the negative control.   3. File Piezo_homologs.faa contains 1229 non-redundant homologs of the Piezo family.</p

Comparative population genomic analyses of transporters within the Asgard archaeal superphylum.

Upon discovery of the first archaeal species in the 1970s, life has been subdivided into three domains: Eukarya, Archaea, and Bacteria. However, the organization of the three-domain tree of life has been challenged following the discovery of archaeal lineages such as the TACK and Asgard superphyla. The Asgard Superphylum has emerged as the closest archaeal ancestor to eukaryotes, potentially improving our understanding of the evolution of life forms. We characterized the transportomes and their substrates within four metagenome-assembled genomes (MAGs), that is, Odin-, Thor-, Heimdall- and Loki-archaeota as well as the fully sequenced genome of Candidatus Prometheoarchaeum syntrophicum strain MK-D1 that belongs to the Loki phylum. Using the Transporter Classification Database (TCDB) as reference, candidate transporters encoded within the proteomes were identified based on sequence similarity, alignment coverage, compatibility of hydropathy profiles, TMS topologies and shared domains. Identified transport systems were compared within the Asgard superphylum as well as within dissimilar eukaryotic, archaeal and bacterial organisms. From these analyses, we infer that Asgard organisms rely mostly on the transport of substrates driven by the proton motive force (pmf), the proton electrochemical gradient which then can be used for ATP production and to drive the activities of secondary carriers. The results indicate that Asgard archaea depend heavily on the uptake of organic molecules such as lipid precursors, amino acids and their derivatives, and sugars and their derivatives. Overall, the majority of the transporters identified are more similar to prokaryotic transporters than eukaryotic systems although several instances of the reverse were documented. Taken together, the results support the previous suggestions that the Asgard superphylum includes organisms that are largely mixotrophic and anaerobic but more clearly define their metabolic potential while providing evidence regarding their relatedness to eukaryotes

Piezo_Suppl_File_S1

This file contains a directory with 10 Multiple sequence alignment (MSA) files in clustal format. Note that all alignments were trimmed by removing positions with more than 30% gaps (see Methods): 1. Piezo.aln contains the MSA of the 279 full length proteins that were used in our analysis.  2. Rep1.aln to Rep9.aln contain MSAs for each individual repeat. Sequences that did not cover at least 80% of the corresponding repeat were removed (some Piezo members do not contain all 9 repeats).</p

Average hydropathy plot (dark red line) showing the basis for the topological predictions made for the <i>Nectria haematococca (Fusarium solani)</i> nhTMEM16 (anoctamin) protein (TC: 1.A.17.1.18), for which x-ray structures are available (PDB IDs 4WIS and 4WIT).

<p>Vertical tan bars show the positions of the predicted TMSs using the Loop Finder program (V. S. Reddy and M. H. Saier, unpublished). The green bar shows the position of the α-helix corresponding to TMS 6. This helix was not predicted to be a TMS by this program, HMMTOP [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192851#pone.0192851.ref096" target="_blank">96</a>] or CCTOP [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192851#pone.0192851.ref108" target="_blank">108</a>], although the x-ray structure confirmed that it is one. HMMTOP predicted TMSs 1 and 2 as a single TMS, although the structure confirms that the corresponding hydrophobicity peak is composed of two TMSs. The two purple bars, representing the position of transmembrane helices 7 and 8 in the x-ray structure, were predicted by these programs and AveHAS [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192851#pone.0192851.ref106" target="_blank">106</a>] to be a single TMS (also note the 7^th hydrophobicity peak in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192851#pone.0192851.g004" target="_blank">Fig 4A</a>). This explains the discrepancy in the predictions for different members of the Anoctamin Superfamily (between 8 and 10 TMSs). The locations, in the hydropathy curve, of the three pairs of functional residues that bind Ca²⁺ in TMSs 6, 7 and 8 are depicted with blue, black and green circles, respectively.</p

Average topological features of the seven families within the Anoctamin Superfamily.

<p>Plots for all families were generated with the AveHAS [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192851#pone.0192851.ref106" target="_blank">106</a>] program. Each plot is composed of two curves. Top dark red lines represent average hydropathy. Bottom gray dotted lines represent average similarity. Predicted TMSs are shown as vertical gray lines. Numbered bars above the hydropathy curves indicate the positions of peaks of hydrophobicity, usually predicted to be TMSs using the HMMTOP [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192851#pone.0192851.ref096" target="_blank">96</a>] and WHAT [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192851#pone.0192851.ref095" target="_blank">95</a>] programs. This figure shows that there are 8 to 10 hydrophobicity peaks in all seven families, which likely correspond to 9 or 10 TMS, since, in this superfamily, some hydrophobicity peaks (such as peak 7 in <b>A</b>) are composed of 2 TMSs. The similarity curves indicate that the regions containing TMSs have the highest levels of conservation, and the corresponding multiple alignments shows that they have fewer gaps.</p