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Pendrin (SLC26A4) is an anion exchanger that mediates bicarbonate (HCO 3 − ) exchange for chloride (Cl − ) and is crucial for maintaining pH and salt homeostasis in the kidney, lung, and cochlea. Pendrin also exports iodide (I − ) in the thyroid gland. Pendrin mutations in humans lead to Pendred syndrome, causing hearing loss and goiter. Inhibition of pendrin is a validated approach for attenuating airway hyperresponsiveness in asthma and for treating hypertension. However, the mechanism of anion exchange and its inhibition by drugs remains poorly understood. We applied cryo-electron microscopy to determine structures of pendrin from Sus scrofa in the presence of either Cl − , I − , HCO 3 − or in the apo-state. The structures reveal two anion-binding sites in each protomer, and functional analyses show both sites are involved in anion exchange. The structures also show interactions between the Sulfate Transporter and Anti-Sigma factor antagonist (STAS) and transmembrane domains, and mutational studies suggest a regulatory role. We also determine the structure of pendrin in a complex with niflumic acid (NFA), which uncovers a mechanism of inhibition by competing with anion binding and impeding the structural changes necessary for anion exchange. These results reveal directions for understanding the mechanisms of anion selectivity and exchange and their regulations by the STAS domain. This work also establishes a foundation for analyzing the pathophysiology of mutations associated with Pendred syndrome. Here the authors report structures of pendrin, an anion exchanger, in complex with its substrate Cl − , I − , or HCO 3 − , which reveal two anion binding sites in each protomer. The authors also identify binding sites of a pendrin inhibitor, niflumic acid.

Na + /H + antiporters comprise a family of membrane proteins evolutionarily conserved in all kingdoms of life that are essential in cellular ion homeostasis. While several human homologues have long been drug targets, NhaA of Escherichia coli has become the paradigm for this class of secondary active transporters as NhaA crystals provided insight in the structure of this molecular machine. However, structural data revealing the composition of the binding site for Na + (or its surrogate Li + ) is missing, representing a bottleneck in our understanding of the correlation between the structure and function of NhaA. Here, by adapting the scintillation proximity assay (SPA) for direct determination of Na + binding to NhaA, we revealed that (i) NhaA is well adapted as the main antiporter for Na + homeostasis in Escherichia coli and possibly in other bacteria as the cytoplasmic Na + concentration is similar to the Na + binding affinity of NhaA, (ii) experimental conditions affect NhaA-mediated cation binding, (iii) in addition to Na + and Li + , the halide Tl + interacts with NhaA, (iv) whereas acidic pH inhibits maximum binding of Na + to NhaA, partial Na + binding by NhaA is independent of the pH, an important novel insight into the effect of pH on NhaA cation binding.

Secondary active transporters, which are vital for a multitude of physiological processes, use the energy of electrochemical ion gradients to power substrate transport across cell membranes 1 , 2 . Efforts to investigate their mechanisms of action have been hampered by their slow transport rates and the inherent limitations of ensemble methods. Here we quantify the activity of individual MhsT transporters, which are representative of the neurotransmitter:sodium symporter family of secondary transporters 3 , by imaging the transport of individual substrate molecules across lipid bilayers at both single- and multi-turnover resolution. We show that MhsT is active only when physiologically oriented and that the rate-limiting step of the transport cycle varies with the nature of the transported substrate. These findings are consistent with an extracellular allosteric substrate-binding site that modulates the rate-limiting aspects of the transport mechanism 4 , 5 , including the rate at which the transporter returns to an outward-facing state after the transported substrate is released. Imaging of substrate transport by individual MhsT transporters, members of the neurotransmitter:sodium symporter family of secondary transporters, at single- and multi-turnover resolution reveals that the rate-limiting step varies with the identity of the transported substrate.

Anion Exchanger 1 (AE1) is an elevator-type transporter that plays a key role in acid-base homeostasis of erythrocytes. Here, we report three high-resolution cryo-electron microscopy (cryo-EM) structures of distinct states of AE1: two inward-facing (IF1 and IF2) and one outward-facing (OF). Uptake assay revealed the modulatory effect of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) lipids on AE1. Molecular dynamics simulations are conducted on these structures to determine the anion binding sites in AE1. We then use advanced enhanced sampling to study the OF⇌IF transition in AE1 in three systems: apo , HCO 3 – -bound, and an AE1 system in which cryo-EM-determined PIP 2 lipids had been removed. The transition pathways were then used to calculate the free energy of the OF⇌IF transition in AE1 under different conditions. The results show how substrate reduces the transition barrier against transport. Furthermore, they capture the inhibitory effect of PIP 2 lipids and provide a molecular mechanism for this inhibitory effect. AE1 is the most abundant membrane protein in erythrocytes. Here, authors report cryo-EM structures in multiple states and demonstrate the inhibitory effect of PIP2 lipids. Free energy calculations reveal the molecular mechanism for PIP 2 inhibition.

LeuT is a Na + -coupled amino acid transporter that is similar in sequence and function to eukaryotic neurotransmitter/sodium symporters, which are active in reuptake of neurotransmitters from the synapse. Distance measurements between spin-label pairs are used to identify ligand-dependent structural transitions in LeuT. The leucine transporter (LeuT) from Aquifex aeolicus is a bacterial homolog of neurotransmitter/sodium symporters (NSSs) that catalyze reuptake of neurotransmitters at the synapse. Crystal structures of wild-type and mutants of LeuT have been interpreted as conformational states in the coupled transport cycle. However, the mechanistic identities inferred from these structures have not been validated, and the ligand-dependent conformational equilibrium of LeuT has not been defined. Here, we used distance measurements between spin-label pairs to elucidate Na + - and leucine-dependent conformational changes on the intracellular and extracellular sides of the transporter. The results identify structural motifs that underlie the isomerization of LeuT between outward-facing, inward-facing and occluded states. The conformational changes reported here present a dynamic picture of the alternating-access mechanism of LeuT and NSSs that is different from the inferences reached from currently available structural models.

Inhibitors of the bile acid transporter ASBT may be useful therapeutics for treating hypercholesterolaemia and type 2 diabetes; here, two X-ray crystal structures of an ASBT homologue from Yersinia frederiksenii are solved. A model of intestinal bile acid transport This paper reports two X-ray crystal structures of a bacterial homologue of the human apical sodium-dependent bile salt transporter (ASBT, also known as SLC10A2), one of two transporters involved in retrieving secreted bile acids from the intestine. The homologue (termed ASBT Yf ), from Yersinia frederiksenii , was crystallized in a lipid environment. The structures reveal that a large rigid-body rotation of a substrate-binding domain gives alternate accessibility to the highly conserved 'crossover' region, where two discontinuous transmembrane helices cross each other. This result has implications for the location and orientation of the bile acid during transport, as well as for the translocation pathway for sodium ions. The authors cite evidence that implies that overall fold and transport mechanism are similar between ASBT and ASBT Yf and they suggest that ASBT Yf may serve as a useful model system for understanding mechanisms of transport and inhibition in the mammalian ASBT homologues. ASBT inhibitors are being studied as potential therapeutics for the treatment of hypercholesterolaemia and type II diabetes. Bile acids are synthesized from cholesterol in hepatocytes and secreted through the biliary tract into the small intestine, where they aid in absorption of lipids and fat-soluble vitamins. Through a process known as enterohepatic recirculation, more than 90% of secreted bile acids are then retrieved from the intestine and returned to the liver for resecretion 1 . In humans, there are two Na + -dependent bile acid transporters involved in enterohepatic recirculation, the Na + -taurocholate co-transporting polypeptide (NTCP; also known as SLC10A1) expressed in hepatocytes, and the apical sodium-dependent bile acid transporter (ASBT; also known as SLC10A2) expressed on enterocytes in the terminal ileum 2 . In recent years, ASBT has attracted much interest as a potential drug target for treatment of hypercholesterolaemia, because inhibition of ASBT reduces reabsorption of bile acids, thus increasing bile acid synthesis and consequently cholesterol consumption 3 , 4 . However, a lack of three-dimensional structures of bile acid transporters hampers our ability to understand the molecular mechanisms of substrate selectivity and transport, and to interpret the wealth of existing functional data 2 , 5 , 6 , 7 , 8 . The crystal structure of an ASBT homologue from Neisseria meningitidis (ASBT NM ) in detergent was reported recently 9 , showing the protein in an inward-open conformation bound to two Na + and a taurocholic acid. However, the structural changes that bring bile acid and Na + across the membrane are difficult to infer from a single structure. To understand the structural changes associated with the coupled transport of Na + and bile acids, here we solved two structures of an ASBT homologue from Yersinia frederiksenii (ASBT Yf ) in a lipid environment, which reveal that a large rigid-body rotation of a substrate-binding domain gives the conserved ‘crossover’ region, where two discontinuous helices cross each other, alternating accessibility from either side of the cell membrane. This result has implications for the location and orientation of the bile acid during transport, as well as for the translocation pathway for Na + .

Transport proteins constitute [almost equal to]10% of most proteomes and play vital roles in the translocation of solutes across membranes of all organisms. Their (dys)function is implicated in many disorders, making them frequent targets for pharmacotherapy. The identification of substrates for members of this large protein family, still replete with many orphans of unknown function, has proven difficult, in part because high-throughput screening is greatly complicated by endogenous transporters present in many expression systems. In addition, direct structural studies require that transporters be extracted from the membrane with detergent, thereby precluding transport measurements because of the lack of a vectorial environment and necessitating reconstitution into proteoliposomes for activity measurements. Here, we describe a direct scintillation proximity-based radioligand-binding assay for determining transport protein function in crude cell extracts and in purified form. This rapid and universally applicable assay with advantages over cell-based platforms will greatly facilitate the identification of substrates for many orphan transporters and allows monitoring the function of transport proteins in a nonmembranous environment.

Human calcium-sensing receptor (CaSR) is a G-protein-coupled receptor (GPCR) that maintains extracellular Ca2+ homeostasis through the regulation of parathyroid hormone secretion. It functions as a disulfide-tethered homodimer composed of three main domains, the Venus Flytrap module, cysteine-rich domain, and seven-helix transmembrane region. Here, we present the crystal structures of the entire extracellular domain of CaSR in the resting and active conformations. We provide direct evidence that L-amino acids are agonists of the receptor. In the active structure, L-Trp occupies the orthosteric agonist-binding site at the interdomain cleft and is primarily responsible for inducing extracellular domain closure to initiate receptor activation. Our structures reveal multiple binding sites for Ca2+ and PO43- ions. Both ions are crucial for structural integrity of the receptor. While Ca2+ ions stabilize the active state, PO43- ions reinforce the inactive conformation. The activation mechanism of CaSR involves the formation of a novel dimer interface between subunits. Calcium ions regulate many processes in the human body. The calcium-sensing receptor, called CaSR, is responsible for maintaining a stable level of calcium ions in the blood. This receptor can detect small changes in the concentration of calcium ions, and activates signalling events within the cell to restore the level of calcium ions back to normal. Abnormal activity of this receptor is associated with severe diseases in humans CaSR is found in the surface membrane of cells and belongs to a family of proteins called G-protein coupled receptors. Much of the protein extends out of the cell and interacts with calcium ions, phosphate ions and certain other molecules such as amino acids. However, it was not well understood how these small molecules bind to CaSR and how this activates the receptor. Geng et al. have now used a technique called X-ray crystallography to view the three-dimensional structure of the exterior domain of CaSR in its resting state and active state. These structures revealed that, contrary to expectations, calcium ions are not the main activator of the receptor. Instead, Geng et al. found that CaSR adopts an inactive state in the absence or presence of calcium ions, while the active state only forms when an amino acid is bound. Furthermore investigation showed that calcium ions are needed to stabilise the active form, while phosphate ions keep the inactive form stable. Geng et al. also identified the shape changes that must occur as CaSR transitions from its inactive to its active state. In particular, an amino acid binding to the exterior domain causes it to close like a venus flytrap, which is a crucial step in activating the receptor. Taken together, the findings show that the amino acids and calcium ions act jointly to fully activate CaSR. The next steps are to determine the structure of the entire receptor with and without its small molecule partners and to use these structures to design drugs that can alter CaSR’s activity in order to treat human diseases.

An open gate to neurotransmitters Neurotransmitters are removed from the synapse by neurotransmitter/Na + symporters (NSSs) in a reuptake process driven by the Na + gradient. Zhao et al . examined the coupling of substrate binding to structural transitions in the prokaryotic NSS homologue LeuT. Using single-molecule fluorescence resonance energy transfer, functional experiments and computational studies, they find that LeuT facilitates intracellular gate opening and substrate release at the intracellular face of the protein. Neurotransmitter/Na + symporters (NSSs) terminate neuronal signalling by recapturing neurotransmitter released into the synapse in a co-transport (symport) mechanism driven by the Na + electrochemical gradient 1 , 2 , 3 , 4 , 5 , 6 . NSSs for dopamine, noradrenaline and serotonin are targeted by the psychostimulants cocaine and amphetamine 1 , as well as by antidepressants 7 . The crystal structure of LeuT, a prokaryotic NSS homologue, revealed an occluded conformation in which a leucine (Leu) and two Na + are bound deep within the protein 8 . This structure has been the basis for extensive structural and computational exploration of the functional mechanisms of proteins with a LeuT-like fold 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 . Subsequently, an ‘outward-open’ conformation was determined in the presence of the inhibitor tryptophan 23 , and the Na + -dependent formation of a dynamic outward-facing intermediate was identified using electron paramagnetic resonance spectroscopy 24 . In addition, single-molecule fluorescence resonance energy transfer imaging has been used to reveal reversible transitions to an inward-open LeuT conformation, which involve the movement of transmembrane helix TM1a away from the transmembrane helical bundle 22 . We investigated how substrate binding is coupled to structural transitions in LeuT during Na + -coupled transport. Here we report a process whereby substrate binding from the extracellular side of LeuT facilitates intracellular gate opening and substrate release at the intracellular face of the protein. In the presence of alanine, a substrate that is transported ∼10-fold faster than leucine 15 , 25 , we observed alanine-induced dynamics in the intracellular gate region of LeuT that directly correlate with transport efficiency. Collectively, our data reveal functionally relevant and previously hidden aspects of the NSS transport mechanism that emphasize the functional importance of a second substrate (S2) binding site within the extracellular vestibule 15 , 20 . Substrate binding in this S2 site appears to act cooperatively with the primary substrate (S1) binding site to control intracellular gating more than 30 Å away, in a manner that allows the Na + gradient to power the transport mechanism.

Enzymes undergo dynamic conformational changes during catalysis, yet conventional high-resolution structural methods typically capture only the most stable states. Here, we address this gap using rapid UV photolysis of a chemically caged substrate with cryogenic time-resolved electron microscopy (cryo-TREM). We elucidate the catalytic mechanism of GtrB, a membrane-bound glycosyltransferase that transfers glucose from UDP-glucose to the lipid carrier undecaprenyl phosphate. We visualized how GtrB, which has an active site ~15 Å from the membrane, transitions during the catalytic cycle to move each substrate in proximity for catalysis. From a single dataset, we resolved distinct conformational states: the initial substrate-bound state, a catalytically poised intermediate, and the product-bound state. Through molecular dynamics simulations and biochemical analyses, we identify coordinated movements within the active site that drive catalysis. These findings provide a molecular framework for understanding how glycosyltransferases function and highlight a broadly applicable strategy for capturing dynamic enzymatic states in native-like environments. Here the authors applied cryogenic time-resolved electron microscopy with rapid UV photolysis of a caged substrate to elucidate the catalytic mechanism of lipid-sugar transfer within the bacterial membrane by the glycosyltransferase GtrB.