Structural basis of promiscuous substrate transport by Organic Cation Transporter 1.

Organic Cation Transporter 1 (OCT1) plays a crucial role in hepatic metabolism by mediating the uptake of a range of metabolites and drugs. Genetic variations can alter the efficacy and safety of compounds transported by OCT1, such as those used for cardiovascular, oncological, and psychological indications. Despite its importance in drug pharmacokinetics, the substrate selectivity and underlying structural mechanisms of OCT1 remain poorly understood. Here, we present cryo-EM structures of full-length human OCT1 in the inward-open conformation, both ligand-free and drug-bound, indicating the basis for its broad substrate recognition. Comparison of our structures with those of outward-open OCTs provides molecular insight into the alternating access mechanism of OCTs. We observe that hydrophobic gates stabilize the inward-facing conformation, whereas charge neutralization in the binding pocket facilitates the release of cationic substrates. These findings provide a framework for understanding the structural basis of the promiscuity of drug binding and substrate translocation in OCT1.

OCTN1: A Widely Studied but Still Enigmatic Organic Cation Transporter Linked to Human Pathology and Drug Interactions.

The Novel Organic Cation Transporter, OCTN1, is the first member of the OCTN subfamily; it belongs to the wider Solute Carrier family SLC22, which counts many members including cation and anion organic transporters. The tertiary structure has not been resolved for any cation organic transporter. The functional role of OCNT1 is still not well assessed despite the many functional studies so far conducted. The lack of a definitive identification of OCTN1 function can be attributed to the different experimental systems and methodologies adopted for studying each of the proposed ligands. Apart from the contradictory data, the international scientific community agrees on a role of OCTN1 in protecting cells and tissues from oxidative and/or inflammatory damage. Moreover, the involvement of this transporter in drug interactions and delivery has been well clarified, even though the exact profile of the transported/interacting molecules is still somehow confusing. Therefore, OCTN1 continues to be a hot topic in terms of its functional role and structure. This review focuses on the most recent advances on OCTN1 in terms of functional aspects, physiological roles, substrate specificity, drug interactions, tissue expression, and relationships with pathology.

Crystal structures of a nicotine MATE transporter provide insight into its mechanism of substrate transport.

A transporter of the multidrug and toxic compound extrusion (MATE) family, Nicotiana tabacum MATE2 (NtMATE2), is located in the vacuole membrane of the tobacco plant root and is involved in the transportation of nicotine, a secondary or specialized metabolic compound in Solanaceae. Here, we report the crystal structures of NtMATE2 in its outward-facing forms. The overall structure has a bilobate V-shape with pseudo-symmetrical assembly of the N- and C-lobes. In one crystal structure, the C-lobe cavity of NtMATE2 interacts with an unidentified molecule that may partially mimic a substrate. In addition, NtMATE2-specific conformational transitions imply that an unprecedented movement of the transmembrane α-helix 7 is related to the release of the substrate into the vacuolar lumen.

Involvement of Human Multidrug and Toxic Compound Extrusion (MATE) Transporters in Testosterone Transport.

Multidrug and toxic compound extrusion (MATE) transporters are primarily expressed in the kidneys and liver, where they contribute to the excretion of organic cations. Our previous study suggested that pig MATE2 (class III) participates in testosterone secretion from Leydig cells. In humans, it is unclear which MATE class is involved in testosterone transport. In this study, we aimed to clarify whether human MATE1 (hMATE1) or human MATE2K (hMATE2K) mediates testosterone transport. To confirm that testosterone inhibits transporter-mediated tetraethylammonium (TEA) uptake, a cis-inhibition assay was performed using cells that stably expressed hMATE1 or hMATE2K. Docking simulations were performed to characterize differences in the binding of hMATE1 and hMATE2K to testosterone. Transport experiments in LLC-PK1 cells that stably expressed hMATE1 were used to test whether hMATE1 mediates testosterone transport. We detected differences between the amino acid sequences of the substrate-binding sites of hMATE1 and hMATE2K that could potentially be involved in testosterone binding. Testosterone and estradiol inhibited TEA uptake mediated by hMATE1 but not that mediated by hMATE2K. Transport experiments in LLC-PK1 cells indicated that testosterone might be transported via hMATE1. This study suggested that hMATE1, but not hMATE2K, is involved in human testosterone transport.

SLC22A14 is a mitochondrial riboflavin transporter required for sperm oxidative phosphorylation and male fertility.

Ablation of Slc22a14 causes male infertility in mice, but the underlying mechanisms remain unknown. Here, we show that SLC22A14 is a riboflavin transporter localized at the inner mitochondrial membrane of the spermatozoa mid-piece and show by genetic, biochemical, multi-omic, and nutritional evidence that riboflavin transport deficiency suppresses the oxidative phosphorylation and reprograms spermatozoa energy metabolism by disrupting flavoenzyme functions. Specifically, we find that fatty acid ß-oxidation (FAO) is defective with significantly reduced levels of acyl-carnitines and metabolites from the TCA cycle (the citric acid cycle) but accumulated triglycerides and free fatty acids in Slc22a14 knockout spermatozoa. We demonstrate that Slc22a14-mediated FAO is essential for spermatozoa energy generation and motility. Furthermore, sperm from wild-type mice treated with a riboflavin-deficient diet mimics those in Slc22a14 knockout mice, confirming that an altered riboflavin level causes spermatozoa morphological and bioenergetic defects. Beyond substantially advancing our understanding of spermatozoa energy metabolism, our study provides an attractive target for the development of male contraceptives.

Conserved binding site in the N-lobe of prokaryotic MATE transporters suggests a role for Na+ in ion-coupled drug efflux.

In both prokaryotes and eukaryotes, multidrug and toxic-compound extrusion (MATE) transporters catalyze the efflux of a broad range of cytotoxic compounds, including human-made antibiotics and anticancer drugs. MATEs are secondary-active antiporters, i.e., their drug-efflux activity is coupled to, and powered by, the uptake of ions down a preexisting transmembrane electrochemical gradient. Key aspects of this mechanism, however, remain to be delineated, such as its ion specificity and stoichiometry. We previously revealed the existence of a Na+-binding site in a MATE transporter from Pyroccocus furiosus (PfMATE) and hypothesized that this site might be broadly conserved among prokaryotic MATEs. Here, we evaluate this hypothesis by analyzing VcmN and ClbM, which along with PfMATE are the only three prokaryotic MATEs whose molecular structures have been determined at atomic resolution, i.e. better than 3 Å. Reinterpretation of existing crystallographic data and molecular dynamics simulations indeed reveal an occupied Na+-binding site in the N-terminal lobe of both structures, analogous to that identified in PfMATE. We likewise find this site to be strongly selective against K+, suggesting it is mechanistically significant. Consistent with these computational results, DEER spectroscopy measurements for multiple doubly-spin-labeled VcmN constructs demonstrate Na+-dependent changes in protein conformation. The existence of this binding site in three MATE orthologs implicates Na+ in the ion-coupled drug-efflux mechanisms of this class of transporters. These results also imply that observations of H+-dependent activity likely stem either from a site elsewhere in the structure, or from H+ displacing Na+ under certain laboratory conditions, as has been noted for other Na+-driven transport systems.

Principles of Alternating Access in Multidrug and Toxin Extrusion (MATE) Transporters.

The multidrug and toxin extrusion (MATE) transporters catalyze active efflux of a broad range of chemically- and structurally-diverse compounds including antimicrobials and chemotherapeutics, thus contributing to multidrug resistance in pathogenic bacteria and cancers. Multiple methodological approaches have been taken to investigate the structural basis of energy transduction and substrate translocation in MATE transporters. Crystal structures representing members from all three MATE subfamilies have been interpreted within the context of an alternating access mechanism that postulates occupation of distinct structural intermediates in a conformational cycle powered by electrochemical ion gradients. Here we review the structural biology of MATE transporters, integrating the crystallographic models with biophysical and computational studies to define the molecular determinants that shape the transport energy landscape. This holistic analysis highlights both shared and disparate structural and functional features within the MATE family, which underpin an emerging theme of mechanistic diversity within the framework of a conserved structural scaffold.

Choline transporter-like proteins 1 and 2 are newly identified plasma membrane and mitochondrial ethanolamine transporters.

The membrane phospholipids phosphatidylcholine and phosphatidylethanolamine (PE) are synthesized de novo by the CDP-choline and CDP-ethanolamine (Kennedy) pathway, in which the extracellular substrates choline and ethanolamine are transported into the cell, phosphorylated, and coupled with diacylglycerol to form the final phospholipid product. Although multiple transport systems have been established for choline, ethanolamine transport is poorly characterized and there is no single protein assigned a transport function for ethanolamine. The solute carriers 44A (SLC44A) known as choline transporter-like proteins-1 and -2 (CTL1 and CTL2) are choline transporter at the plasma membrane and mitochondria. We report a novel function of CTL1 and CTL2 in ethanolamine transport. Using the lack or the gain of gene function in combination with specific antibodies and transport inhibitors we established two distinct ethanolamine transport systems of a high affinity, mediated by CTL1, and of a low affinity, mediated by CTL2. Both transporters are Na+-independent ethanolamine/H+ antiporters. Primary human fibroblasts with separate frameshift mutations in the CTL1 gene (M1= SLC44A1ΔAsp517 and M2= SLC44A1ΔSer126) are devoid of CTL1 ethanolamine transport but maintain unaffected CTL2 transport. The lack of CTL1 in M2 cells reduced the ethanolamine transport, the flux through the CDP-ethanolamine Kennedy pathway, and PE synthesis. In contrast, overexpression of CTL1 in M2 cells improved ethanolamine transport and PE synthesis. These data firmly establish that CTL1 and CTL2 are the first identified ethanolamine transporters in whole cells and mitochondria, with intrinsic roles in de novo PE synthesis by the Kennedy pathway and intracellular redistribution of ethanolamine.

Baicalein alleviates hyperuricemia by promoting uric acid excretion and inhibiting xanthine oxidase.

BACKGROUND: Insufficient renal urate excretion and/or overproduction of uric acid (UA) are the dominant causes of hyperuricemia. Baicalein (BAL) is widely distributed in dietary plants and has extensive biological activities, including antioxidative, anti-inflammatory and antihypertensive activities. PURPOSE: To investigate the anti-hyperuricemic effects of BAL and the underlying mechanisms in vitro and in vivo. METHODS: We investigated the inhibitory effects of BAL on GLUT9 and URAT1 in vitro through electrophysiological experiments and 14C-urate uptake assays. To evaluate the impact of BAL on serum and urine UA, the expression of GLUT9 and URAT1, and the activity of xanthine oxidase (XOD), we developed a mouse hyperuricemia model by potassium oxonate (PO) injection. Molecular docking analysis based on homology modeling was performed to explain the predominant efficacy of BAL compared with the other test compounds. RESULTS: BAL dose-dependently inhibited GLUT9 and URAT1 in a noncompetitive manner with IC50 values of 30.17 ± 8.68 µM and 31.56 ± 1.37 µM, respectively. BAL (200 mg/kg) significantly decreased serum UA and enhanced renal urate excretion in PO-induced hyperuricemic mice. Moreover, the expression of GLUT9 and URAT1 in the kidney was downregulated, and XOD activity in the serum and liver was suppressed. The docking analysis revealed that BAL potently interacted with Trp336, Asp462, Tyr71 and Gln328 of GLUT9 and Ser35 and Phe241 of URAT1. CONCLUSION: These results indicated that BAL exerts potent antihyperuricemic efects through renal UA excretal promotion and serum UA production. Thus, we propose that BAL may be a promising treatment for the prevention of hyperuricemia owing to its multitargeted inhibitory activity.

Sequence and structural determinants of ligand-dependent alternating access of a MATE transporter.

Multidrug and toxic compound extrusion (MATE) transporters are ubiquitous ion-coupled antiporters that extrude structurally and chemically dissimilar cytotoxic compounds and have been implicated in conferring multidrug resistance. Here, we integrate double electron-electron resonance (DEER) with functional assays and site-directed mutagenesis of conserved residues to illuminate principles of ligand-dependent alternating access of PfMATE, a proton-coupled MATE from the hyperthermophilic archaeon Pyrococcus furiosus Pairs of spin labels monitoring the two sides of the transporter reconstituted into nanodiscs reveal large-amplitude movement of helices that alter the orientation of a putative substrate binding cavity. We found that acidic pH favors formation of an inward-facing (IF) conformation, whereas elevated pH (>7) and the substrate rhodamine 6G stabilizes an outward-facing (OF) conformation. The lipid-dependent PfMATE isomerization between OF and IF conformation is driven by protonation of a previously unidentified intracellular glutamate residue that is critical for drug resistance. Our results can be framed in a mechanistic model of transport that addresses central aspects of ligand coupling and alternating access.

Assessment of Interaction of Human OCT 1-3 Proteins and Metformin Using Silico Analyses.

Metformin, a drug frequently used by diabetic patients as the first-line treatment worldwide, is positively charged and is transported into the cell through human organic cation transporter (hOCT 1-3) proteins. We aimed to mimic the cellular uptake of metformin by hOCT1-3 with various bioinformatics methods and tools. 3D structure of OCT1-3 proteins was predicted by considering the structures and function of these proteins. We predicted functional regions (active and ligand binding sites) of OCT1-3 and performed comparative bioinformatics analysis. The predicted structure of hOCT1-3 was then analyzed in the Blind Docking server and the results were confirmed with predicted binding site residues and conserved domain regions. We simulated the OCT1-3 and metformin docking and also validated the docking procedure with other substrates of HOCT1-3 proteins. We selected the best poses of metformin docking simulations as per binding energy (-5.27 to -4.60 kcal/mol). Lastly, we validated the static description of protein-ligand (OCT-Metformin) interactions by performing molecular dynamics simulation. Eventually, we obtained stable simulation of OCT-metformin interaction.

Two- and three-dimensional QSAR studies on hURAT1 inhibitors with flexible linkers: topomer CoMFA and HQSAR.

hURAT1 (human urate transporter 1) is a successful target for hyperuricemia. Recently, the modification work on hURAT1 inhibitors showed that the flexible linkers would benefit biological activity. The study aimed to investigate the contribution of the linkers and give modification strategies on this kind of structures based on QSAR models (HQSAR and topomer CoMFA). The most effective HQSAR and topomer CoMFA models were generated by applying the training set containing 63 compounds, with the cross-validated q2 values of 0.869/0.818 and the non-cross-validated correlation coefficients r2 of 0.951/0.978, respectively. The Y-randomization test was applied to ensure the robustness of the models. The external predictive correlation coefficient (rpred2) grounded on the external test set (21 compounds) of two models was 0.910 and 0.907, respectively. In addition, the models were validated by Golbraikh-Tropsha and Roy methods, as well as other statistical metrics. The results showed that both models were reliable. Topomer CoMFA steric/electrostatic contours and HQSAR atomic contribution maps illustrated the structural features which governed their inhibitory potency. The dependable results could provide important insights to guide the designing of more potential hURAT1 inhibitors.

On the ion coupling mechanism of the MATE transporter ClbM.

Bacteria use a number of mechanisms to defend themselves from antimicrobial drugs. One important defense strategy is the ability to export drugs by multidrug transporters. One class of multidrug transporter, the so-called multidrug and toxic compound extrusion (MATE) transporters, extrude a variety of antibiotic compounds from the bacterial cytoplasm. These MATE transporters are driven by a Na+, H+, or combined Na+/H+ gradient, and act as antiporters to drive a conformational change in the transporter from the outward to the inward-facing conformation. In the inward-facing conformation, a chemical compound (drug) binds to the protein, resulting in a switch to the opposite conformation, thereby extruding the drug. Using molecular dynamics simulations, we now report the structural basis for Na+ and H+ binding in the dual ion coupled MATE transporter ClbM from Escherichia coli, which is connected to colibactin-induced genotoxicity, yielding novel insights into the ion/drug translocation mechanism of this bacterial transporter.

Structural biology of the multidrug and toxic compound extrusion superfamily transporters.

Xenobiotic and metabolite extrusion is an important process for the proper functions of cells and their compartments, including acidic organelles. MATE (multidrug and toxic compound extrusion) is a large family of secondary active transporters involved in the transport of various compounds across cellular and organellar membranes, and is present in the three domains of life. The major substrates of the bacterial MATE transporters are cationic compounds, including clinically important antibiotics, and thereby MATE transporters confer multi-drug resistance to pathogenic bacteria. The plant MATE transporters are important for the accumulation of various metabolites in organelles, including vacuoles. The human MATE transporters are expressed in the brush-border membrane of the kidney, and are involved in the clearance of cationic drugs from the body. During the past decade, progress in structural biology has clarified the transport mechanism of these MATE transporters in atomic detail. The present review summarizes the reported structures of MATE family transporters, along with their structure-guided functional analyses. This integrated view of the structures of MATE transporters provides novel insights into their transport mechanism.

Unraveling the functional role of the orphan solute carrier, SLC22A24 in the transport of steroid conjugates through metabolomic and genome-wide association studies.

Variation in steroid hormone levels has wide implications for health and disease. The genes encoding the proteins involved in steroid disposition represent key determinants of interindividual variation in steroid levels and ultimately, their effects. Beginning with metabolomic data from genome-wide association studies (GWAS), we observed that genetic variants in the orphan transporter, SLC22A24 were significantly associated with levels of androsterone glucuronide and etiocholanolone glucuronide (sentinel SNPs p-value <1x10-30). In cells over-expressing human or various mammalian orthologs of SLC22A24, we showed that steroid conjugates and bile acids were substrates of the transporter. Phylogenetic, genomic, and transcriptomic analyses suggested that SLC22A24 has a specialized role in the kidney and appears to function in the reabsorption of organic anions, and in particular, anionic steroids. Phenome-wide analysis showed that functional variants of SLC22A24 are associated with human disease such as cardiovascular diseases and acne, which have been linked to dysregulated steroid metabolism. Collectively, these functional genomic studies reveal a previously uncharacterized protein involved in steroid homeostasis, opening up new possibilities for SLC22A24 as a pharmacological target for regulating steroid levels.

ECOD: identification of distant homology among multidomain and transmembrane domain proteins.

The manual classification of protein domains is approaching its 20th anniversary. ECOD is our mixed manual-automatic domain classification. Over time, the types of proteins which require manual curation has changed. Depositions with complex multidomain and multichain arrangements are commonplace. Transmembrane domains are regularly classified. Repeatedly, domains which are initially believed to be novel are found to have homologous links to existing classified domains. Here we present a brief summary of recent manual curation efforts in ECOD generally combined with specific case studies of transmembrane and multidomain proteins wherein manual curation was useful for discovering new homologous relationships. We present a new taxonomy for the classification of ABC transporter transmembrane domains. We examine alternate topologies of the leucine-specific (LS) domain of Leucine tRNA-synthetase. Finally, we elaborate on a distant homologous links between two helical dimerization domains.

Interaction of Alt a 1 with SLC22A17 in the airway mucosa.

BACKGROUND: Despite all the efforts made up to now, the reasons that facilitate a protein becoming an allergen have not been elucidated yet. Alt a 1 protein is the major fungal allergen responsible for chronic asthma, but little is known about its immunological activity. Our main purpose was to investigate the ligand-dependent interactions of Alt a 1 in the human airway epithelium. METHODS: Alt a 1 with and without its ligand (holo- and apo- forms) was incubated with the pulmonary epithelial monolayer model, Calu-3 cells. Allergen transport and cytokine production were measured. Pull-down and immunofluorescence assays were employed to identify the receptor of Alt a 1 using the epithelial cell model and mouse tissues. Receptor-allergen-ligand interactions were analyzed by computational modeling. RESULTS: The holo-form could activate human monocytes, PBMCs, and polarized airway epithelial (Calu-3) cell lines. The allergen was also transported through the monolayer, without any alteration of the epithelial integrity (TEER). Alt a 1 also induced the production of proinflammatory IL8 and specific epithelial cytokines (IL33 and IL25) by Calu-3 cells. The interaction between epithelial cells and holo-Alt a 1 was found to be mediated by the SLC22A17 receptor, and its recognition of Alt a 1 was explained in structural terms. CONCLUSIONS: Our findings identified the Alt a 1 ligand as a central player in the interaction of the allergen with airway mucosa, shedding light into its potential role in the immunological response, while unveiling its potential as a new target for therapy intervention.

Structural Basis of H+-Dependent Conformational Change in a Bacterial MATE Transporter.

Multidrug and toxic compound extrusion (MATE) transporters efflux toxic compounds using a Na+ or H+ gradient across the membrane. Although the structures of MATE transporters have been reported, the cation-coupled substrate transport mechanism remains controversial. Here we report crystal structures of VcmN, a Vibrio cholerae MATE transporter driven by the H+ gradient. High-resolution structures in two distinct conformations associated with different pHs revealed that the rearrangement of the hydrogen-bonding network around the conserved Asp35 induces the bending of transmembrane helix 1, as in the case of the H+-coupled Pyrococcus furiosus MATE transporter. We also determined the crystal structure of the D35N mutant, which captured a unique conformation of TM1 facilitated by an altered hydrogen-bonding network. Based on the present results, we propose a common step in the transport cycle shared among prokaryotic H+-coupled MATE transporters.

OCTN: A Small Transporter Subfamily with Great Relevance to Human Pathophysiology, Drug Discovery, and Diagnostics.

OCTN is a small subfamily of membrane transport proteins that belongs to the larger SLC22 family. Two of the three members of the subfamily, namely, OCTN2 and OCTN1, are present in humans. OCTN2 plays a crucial role in the absorption of carnitine from diet and in its distribution to tissues, as demonstrated by the occurrence of severe pathologies caused by malfunctioning or altered expression of this transporter. These findings suggest avoiding a strict vegetarian diet during pregnancy and in childhood. Other roles of OCTN2 are related to the traffic of carnitine derivatives in many tissues. The role of OCTN1 is still unclear, despite the identification of some substrates such as ergothioneine, acetylcholine, and choline. Plausibly, the transporter acts on the control of inflammation and oxidative stress, even though knockout mice do not display phenotypes. A clear role of both transporters has been revealed in drug interaction and delivery. The polyspecificity of the OCTNs is at the base of the interactions with drugs. Interestingly, OCTN2 has been recently exploited in the prodrug approach and in diagnostics. A promising application derives from the localization of OCTN2 in exosomes that represent a noninvasive diagnostic tool.

Large-scale whole-exome sequencing association studies identify rare functional variants influencing serum urate levels.

Elevated serum urate levels can cause gout, an excruciating disease with suboptimal treatment. Previous GWAS identified common variants with modest effects on serum urate. Here we report large-scale whole-exome sequencing association studies of serum urate and kidney function among ≤19,517 European ancestry and African-American individuals. We identify aggregate associations of low-frequency damaging variants in the urate transporters SLC22A12 (URAT1; p = 1.3 × 10-56) and SLC2A9 (p = 4.5 × 10-7). Gout risk in rare SLC22A12 variant carriers is halved (OR = 0.5, p = 4.9 × 10-3). Selected rare variants in SLC22A12 are validated in transport studies, confirming three as loss-of-function (R325W, R405C, and T467M) and illustrating the therapeutic potential of the new URAT1-blocker lesinurad. In SLC2A9, mapping of rare variants of large effects onto the predicted protein structure reveals new residues that may affect urate binding. These findings provide new insights into the genetic architecture of serum urate, and highlight molecular targets in SLC22A12 and SLC2A9 for lowering serum urate and preventing gout.