Structural insight into the substrate recognition and transport mechanism of amino acid transporter complex ACE2-B0AT1 and ACE2-SIT1.
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ABSTRACT:
SUBMITTER: Li Y
PROVIDER: S-EPMC10491607 | biostudies-literature | 2023 Sep
REPOSITORIES: biostudies-literature
Publications
Structural insight into the substrate recognition and transport mechanism of amino acid transporter complex ACE2-B<sup>0</sup>AT1 and ACE2-SIT1.
Li Yaning Y Chen Yiming Y Zhang Yuanyuan Y Shen Yaping Y Xu Kangtai K Liu Yaqi Y Wang Zilong Z Yan Renhong R
Cell discovery 20230908 1
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Project description:The SARS-CoV-2 receptor, angiotensin-converting enzyme-2 (ACE2), is expressed at levels of greatest magnitude in the small intestine as compared with all other human tissues. Enterocyte ACE2 is coexpressed as the apical membrane trafficking partner obligatory for expression and activity of the B0AT1 sodium-dependent neutral amino acid transporter. These components are assembled as an [ACE2:B0AT1]2 dimer-of-heterodimers quaternary complex that putatively steers SARS-CoV-2 tropism in the gastrointestinal (GI) tract. GI clinical symptomology is reported in about half of COVID-19 patients, and can be accompanied by gut shedding of virion particles. We hypothesized that within this 4-mer structural complex, each [ACE2:B0AT1] heterodimer pair constitutes a physiological "functional unit." This was confirmed experimentally by employing purified lyophilized enterocyte brush border membrane vesicles exposed to increasing doses of high-energy electron radiation from a 16 MeV linear accelerator. Based on radiation target theory, the results indicated the presence of Na+-dependent neutral amino acid influx transport activity functional unit with target size molecular weight 183.7 ± 16.8 kDa in situ in intact apical membranes. Each thermodynamically stabilized [ACE2:B0AT1] heterodimer functional unit manifests the transport activity within the whole ∼345 kDa [ACE2:B0AT1]2 dimer-of-heterodimers quaternary structural complex. The results are consistent with our prior molecular docking modeling and gut-lung axis approaches to understanding COVID-19. These findings advance understanding the physiology of B0AT1 interaction with ACE2 in the gut, and thereby contribute to translational developments designed to treat or mitigate COVID-19 variant outbreaks and/or GI symptom persistence in long-haul postacute sequelae of SARS-CoV-2.
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Project description:Sugar Will Eventually be Exported Transporters (SWEETs) are recently identified sugar transporters that can discriminate and transport di- or monosaccharides across a membrane following the concentration gradient. SWEETs play key roles in plant biological processes, such as pollen nutrition, nectar secretion, seed filling, and phloem loading. SWEET13 from Arabidopsis thaliana (AtSWEET13) is an important sucrose transporter in pollen development. Here, we report the 2.8-Å resolution crystal structure of AtSWEET13 in the inward-facing conformation with a substrate analog, 2'-deoxycytidine 5'-monophosphate, bound in the central cavity. In addition, based on the results of an in-cell transport activity assay and single-molecule Förster resonance energy transfer analysis, we suggest a mechanism for substrate selectivity based on the size of the substrate-binding pocket. Furthermore, AtSWEET13 appears to form a higher order structure presumably related to its function.
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Project description:Heteromeric amino acid transporters (HATs), including y+LAT1-4F2hc complex, are responsible for transporting amino acids across membranes, and mutations in y+LAT1 cause lysinuric protein intolerance (LPI), a hereditary disorder characterized by defective cationic amino acid transport. The relationship between LPI and specific mutations in y+LAT1 has yet to be fully understood. In this study, we characterized the function of y+LAT1-4F2hc complex in mammalian cells and determined the cryo-EM structures of the human y+LAT1-4F2hc complex in two distinct conformations: the apo state in an inward-open conformation and the native substrate-bound state in an outward-open conformation. Structural analysis suggests that Asp243 in y+LAT1 plays a crucial role in coordination with sodium ion and substrate selectivity. Molecular dynamic (MD) simulations further revealed the different transport mechanism of cationic amino acids and neutral amino acids. These results provide important insights into the mechanisms of the substrate binding and working cycle of HATs.
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Project description:The brush-border membrane of the small intestine and kidney proximal tubule are the major sites for the absorption and re-absorption of nutrients in the body respectively. Transport of amino acids is mediated through the action of numerous secondary active transporters. In the mouse, neutral amino acids are transported by B(0)AT1 [broad neutral ((0)) amino acid transporter 1; SLC6A19 (solute carrier family 6 member 19)] in the intestine and by B(0)AT1 and B(0)AT3 (SLC6A18) in the kidney. Immunoprecipitation and Blue native electrophoresis of intestinal brush-border membrane proteins revealed that B(0)AT1 forms complexes with two peptidases, APN (aminopeptidase N/CD13) and ACE2 (angiotensin-converting enzyme 2). Physiological characterization of B(0)AT1 expressed together with these peptidases in Xenopus laevis oocytes revealed that APN increased the substrate affinity of the transporter up to 2.5-fold and also increased its surface expression (V(max)). Peptide competition experiments, in silico modelling and site-directed mutagenesis of APN suggest that the catalytic site of the peptidase is involved in the observed changes of B(0)AT1 apparent substrate affinity, possibly by increasing the local substrate concentration. These results provide evidence for the existence of B(0)AT1-containing digestive complexes in the brush-border membrane, interacting differentially with various peptidases, and responding to the dynamic needs of nutrient absorption in the intestine and kidney.
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Project description:The pharmacology of amino acid transporters in the SLC6 family is poorly developed compared to that of the neurotransmitter transporters. To identify new inhibitors of the proline transporter SIT1 (SLC6A20), its expression in Xenopus laevis oocytes was optimized. Trafficking of SIT1 was augmented by co-expression of angiotensin-converting enzyme 2 (ACE2) in oocytes but there was no strict requirement for co-expression of ACE2. A pharmacophore-guided screen identified tiagabine as a potent non-competitive inhibitor of SIT1. To understand its binding mode, we determined the cryo-electron microscopy (cryo-EM) structure of ACE2-SIT1 bound with tiagabine. The inhibitor binds close to the orthosteric proline binding site, but due to its size extends into the cytosolic vestibule. This causes the transporter to adopt an inward-open conformation, in which the intracellular gate is blocked. This study provides the first structural insight into inhibition of SIT1 and generates tools for a better understanding of the ACE2-SIT1 complex. These findings may have significance for SARS-CoV-2 binding to its receptor ACE2 in human lung alveolar cells where SIT1 and ACE2 are functionally expressed.
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Project description:Background and purposeThe neutral amino acid transporter B0 AT1 (SLC6A19) has recently been identified as a possible target to treat type 2 diabetes and related disorders. B0 AT1 mediates the Na+ -dependent uptake of all neutral amino acids. For surface expression and catalytic activity, B0 AT1 requires coexpression of collectrin (TMEM27). In this study, we established tools to identify and evaluate novel inhibitors of B0 AT1.Experimental approachA CHO-based cell line was generated, stably expressing collectrin and B0 AT1. Using this cell line, a high-throughput screening assay was developed, which uses a fluorescent dye to detect depolarisation of the cell membrane during amino acid uptake via B0 AT1. In parallel to these functional assays, we ran a computational compound screen using AutoDock4 and a homology model of B0 AT1 based on the high-resolution structure of the highly homologous Drosophila dopamine transporter.Key resultsWe characterized a series of novel inhibitors of the B0 AT1 transporter. Benztropine was identified as a competitive inhibitor of the transporter showing an IC50 of 44 ± 9 μM. The compound was selective with regard to related transporters and blocked neutral amino acid uptake in inverted sections of mouse intestine.Conclusion and implicationsThe tools established in this study can be widely used to identify new transport inhibitors. Using these tools, we were able to identify compounds that can be used to study epithelial transport, to induce protein restriction, or be developed further through medicinal chemistry.
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Project description:Using the crystal structure of the uracil transporter UraA of Escherichia coli, we constructed a 3D model of the Aspergillus nidulans uric acid-xanthine/H(+) symporter UapA, which is a prototype member of the Nucleobase-Ascorbate Transporter (NAT) family. The model consists of 14 transmembrane segments (TMSs) divided into a core and a gate domain, the later being distinctly different from that of UraA. By implementing Molecular Mechanics (MM) simulations and quantitative structure-activity relationship (SAR) approaches, we propose a model for the xanthine-UapA complex where the substrate binding site is formed by the polar side chains of residues E356 (TMS8) and Q408 (TMS10) and the backbones of A407 (TMS10) and F155 (TMS3). In addition, our model shows several polar interactions between TMS1-TMS10, TMS1-TMS3, TMS8-TMS10, which seem critical for UapA transport activity. Using extensive docking calculations we identify a cytoplasm-facing substrate trajectory (D360, A363, G411, T416, R417, V463 and A469) connecting the proposed substrate binding site with the cytoplasm, as well as, a possible outward-facing gate leading towards the substrate major binding site. Most importantly, re-evaluation of the plethora of available and analysis of a number of herein constructed UapA mutations strongly supports the UapA structural model. Furthermore, modeling and docking approaches with mammalian NAT homologues provided a molecular rationale on how specificity in this family of carriers might be determined, and further support the importance of selectivity gates acting independently from the major central substrate binding site.
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