The Transporter Classification Database (TCDB; tcdb.org) is a freely accessible reference resource, which provides functional, structural, mechanistic, medical and biotechnological information about transporters from organisms of all types. TCDB is the only transport protein classification database adopted by the International Union of Biochemistry and Molecular Biology (IUBMB) and now (October 1, 2020) consists of 20 653 proteins classified in 15 528 non-redundant transport systems with 1567 tabulated 3D structures, 18 336 reference citations describing 1536 transporter families, of which 26% are members of 82 recognized superfamilies. Overall, this is an increase of over 50% since the last published update of the database in 2016. This comprehensive update of the database contents and features include (i) adoption of a chemical ontology for substrates of transporters, (ii) inclusion of new superfamilies, (iii) a domain-based characterization of transporter families for the identification of new members as well as functional and evolutionary relationships between families, (iv) development of novel software to facilitate curation and use of the database, (v) addition of new subclasses of transport systems including 11 novel types of channels and 3 types of group translocators and (vi) the inclusion of many man-made (artificial) transmembrane pores/channels and carriers.

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.

Sensing and responding to environmental water deficiency and osmotic stresses are essential for the growth, development, and survival of plants. Recently, an osmolality-sensing ion channel called OSCA1 was discovered that functions in sensing hyperosmolality inArabidopsis. Here, we report the cryo-electron microscopy (cryo-EM) structure and function of an OSCA1 homolog from rice (Oryza sativa; OsOSCA1.2), leading to a model of how it could mediate hyperosmolality sensing and transport pathway gating. The structure reveals a dimer; the molecular architecture of each subunit consists of 11 transmembrane (TM) helices and a cytosolic soluble domain that has homology to RNA recognition proteins. The TM domain is structurally related to the TMEM16 family of calcium-dependent ion channels and lipid scramblases. The cytosolic soluble domain possesses a distinct structural feature in the form of extended intracellular helical arms that are parallel to the plasma membrane. These helical arms are well positioned to potentially sense lateral tension on the inner leaflet of the lipid bilayer caused by changes in turgor pressure. Computational dynamic analysis suggests how this domain couples to the TM portion of the molecule to open a transport pathway. Hydrogen/deuterium exchange mass spectrometry (HDXMS) experimentally confirms the conformational dynamics of these coupled domains. These studies provide a framework to understand the structural basis of proposed hyperosmolality sensing in a staple crop plant, extend our knowledge of the anoctamin superfamily important for plants and fungi, and provide a structural mechanism for potentially translating membrane stress to transport regulation.

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1Cryo-EM structure of OSCA1.2 from Oryza sativa: Mechanical basis of 2 hyperosmolality-gating in plants 3 4 5Abstract 46Sensing and responding to environmental water deficiency and osmotic stresses is 47 essential for the growth, development and survival of plants. Recently, sensing ion channel called OSCA1 was discovered that functions in sensing 49 hyperosmolality in Arabidopsis. Here, we report the cryo-EM structure and function of 50 an ion channel from rice (Oryza sativa; OsOSCA1.2), showing how it mediates 51 hyperosmolality sensing and ion permeability. The structure reveals a dimer; the 52 molecular architecture of each subunit consists of eleven transmembrane helices and 53 a cytosolic soluble domain that has homology to RNA recognition proteins. The 54transmembrane domain is structurally related to the TMEM16 family of calcium 55 dependent ion channels and scramblases. The cytosolic soluble domain possesses a 56 distinct structural feature in the form of extended intracellular helical arms that is 57parallel to the plasma membrane. These helical arms are well positioned to sense 58 lateral tension on the inner leaflet of the lipid bilayer caused by changes in turgor 59pressure. Computational dynamic analysis suggests how this domain couples to the 60 transmembrane portion of the molecule to open the channel. Hydrogen-deuterium 61 exchange mass spectrometry (HDXMS) experimentally confirms the conformational 62 dynamics of these coupled domains. The structure provides a framework to 63understand the structural basis of hyperosmolality sensing in an important crop plant, 64 extends our knowledge of the anoctamin superfamily important for plants and fungi, 65and provides a structural mechanism for translating membrane stress to ion transport 66 regulation. 67 68Introduction 69Hyperosmolarity and osmotic stress are among the first physiological responses to 70 changes in salinity and drought. Hyperosmolality triggers increases in cytosolic free 71Ca 2+ concentration and thereby initiates an osmotic stress-induced signal transduction 72 cascade in plants (1-3). Salinity and drought stress trigger diverse protective 73 mechanisms in plants enabling enhanced drought tolerance and reduction of water 74 loss in leaves. 75 76Ion channels have long been hypothesized as sensors of osmotic stress. A candidate 77 membrane protein named OSCA was isolated in a genetic screen for mutants that 78impair the rapid osmotic stress-induced Ca 2+ elevation in plants (1). OSCA1 encodes 79 a multi-spanning membrane protein that functions in osmotic/mechanical stress-80induced activation of ion currents. However, the underlying mechanisms and whether 81OSCA1 itself encodes an ion conducting pore specific for Ca 2+ requires further 82analysis. OSCA1 is a member of a larger gene family in Arabidopsis with 15 members 83(4), and with many homologs encoded in other plants and fungal genomes. 84Furthermore, evolutionary analyses have revealed that OSCA is distantly related to 85 the anoctamin superfamily, that includes the TMEM16 family...

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.

Members of the Piezo family of mechanically activated cation channels are involved in multiple physiological processes including vascular development, cell differentiation, touch perception, hearing and more. Mutations in these proteins are associated with a variety of diseases, such as colorectal adenomatous polyposis, dehydrated hereditary stomacytosis, and hereditary xerocytosis. Available 3D structures for Piezo proteins show nine regions of four transmembrane segments each that have the same fold. Despite the remarkable similarity among the nine characteristic structural repeats in the family, no significant sequence similarity among them has been reported. Using a bioinformatics approach and the Transporter Classification Database (TCDB) as reference, we reliably identified sequence similarity among repeats based on four lines of evidence: (1) HMM-profile similarities across repeats at the family level, (2) pairwise sequence similarities between different repeats across Piezo homologs, (3) Piezo-specific conserved sequence signatures that consistently identify the same regions across repeats, and (4) conserved residues that maintain the same orientation and location in 3D space.

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.