The apical sodium-dependent bile acid transporter (ASBT, SLC10A2) facilitates the enterohepatic circulation of bile salts and plays a key role in cholesterol metabolism. The membrane topology of ASBT was initially scanned using a consensus topography analysis that predominantly predicts a seven transmembrane (TM) domain configuration adhering to the "positive inside" rule. Membrane topology was further evaluated and confirmed by N-glycosylation-scanning mutagenesis, as reporter sites inserted in the putative extracellular loops 1 and 3 were glycosylated. On the basis of a 7TM topology, we built a three-dimensional model of ASBT using an approach of homology-modeling and remote-threading techniques for the extramembranous domains using bacteriorhodopsin as a scaffold for membrane attachment points; the model was refined using energy minimizations and molecular dynamics simulations. Ramachandran scores and other geometric indicators show that the model is comparable in quality to the crystal structures of similar proteins. Simulated annealing and docking of cholic acid, a natural substrate, onto the protein surface revealed four distinct binding sites. Subsequent site-directed mutagenesis of the predicted binding domain further validated the model. This model agrees further with available data for a pathological mutation (P290S) because the mutant model after in silico mutagenesis loses the ability to bind bile acids.

CLC secondary active transporters exchange Cl- for H+. Crystal structures have suggested that the conformational change from occluded to outward-facing states is unusually simple, involving only the rotation of a conserved glutamate (Gluex) upon its protonation. Using 19F NMR, we show that as [H+] is increased to protonate Gluex and enrich the outward-facing state, a residue ~20 Å away from Gluex, near the subunit interface, moves from buried to solvent-exposed. Consistent with functional relevance of this motion, constriction via inter-subunit cross-linking reduces transport. Molecular dynamics simulations indicate that the cross-link dampens extracellular gate-opening motions. In support of this model, mutations that decrease steric contact between Helix N (part of the extracellular gate) and Helix P (at the subunit interface) remove the inhibitory effect of the cross-link. Together, these results demonstrate the formation of a previously uncharacterized 'outward-facing open' state, and highlight the relevance of global structural changes in CLC function.DOI: http://dx.doi.org/10.7554/eLife.11189.001

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Microbial detection requires the recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) that are distributed on the cell surface and within the cytosol. The nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family functions as an intracellular PRR that triggers the innate immune response. The mechanism by which PAMPs enter the cytosol to interact with NLRs, particularly muropeptides derived from the bacterial proteoglycan cell wall, is poorly understood. PEPT2 is a proton-dependent transporter that mediates the active translocation of di-and tripeptides across epithelial tissues, including the lung. Using computational tools, we initially established that bacterial dipeptides, particularly g-D-glutamyl-meso-diaminopimelic acid (g-iE-DAP), are suitable substrates for PEPT2. We then determined in primary cultures of human upper airway epithelia and transiently transfected CHO-PEPT2 cell lines that g-iE-DAP uptake was mediated by PEPT2 with an affinity constant of approximately 193 mM, whereas muramyl dipeptide was not transported. Exposure to g-iE-DAP at the apical surface of differentiated, polarized cultures resulted in activation of the innate immune response in an NOD1-and RIP2-dependent manner, resulting in release of IL-6 and IL-8. Based on these findings we report that PEPT2 plays a vital role in microbial recognition by NLR proteins, particularly with regard to airborne pathogens, thereby participating in host defense in the lung.Keywords: human; lung; bacterial; cell surface molecules; acute phase reactants Pathogen-associated molecular patterns (PAMPs) are recognized by cell surface or cytosolic pattern recognition receptors (PRRs) (1). One family of intracellular PRRs include the nucleotide-binding oligomerization domain (NOD)-containing proteins (NODs or NOD-like receptors [NLRs]) that sense intracellular pathogen invasion and trigger signaling cascades, thereby activating the host immune response (2-4). NOD1 and NOD2 mediate intracellular recognition of bacterial proteoglycan (PGN)-derived molecules through recognition by their carboxy-terminal leucine-rich repeat region (5). This results in recruitment of the downstream kinase protein RIP2 and initiation of NF-kB-mediated transcription and elaboration of newly synthesized cytokines and chemokines (6). Biochemical and functional analyses have identified g-D-glutamyl-mesodiaminopimelic acid (g-iE-DAP) and muramyl dipeptide (MDP) as the minimal bacterial muropeptide epitopes recognized by NOD1 and NOD2, respectively, resulting in immune activation (5, 7).Enzymes that are naturally present in the epithelial lining fluid (e.g., lysozyme) are believed to act in concert to degrade the bacterial cell wall as a front-line defense mechanism, thereby liberating muropeptide fragments that include both g-iE-DAP and MDP. The dipeptides may also be derived as byproducts of bacterial PGN biosynthesis, cell growth, and division; therefore, it is plausible that these particular PAMPs reside in the airway...

The human apical sodium-dependent bile acid transporter (hASBT, SLC10A2) plays a critical role in the enterohepatic circulation of bile acids, as well as in cholesterol homeostasis. ASBT reclaims bile acids from the distal ileum via active sodium co-transport, in a multistep process, orchestrated by key residues in exofacial loop regions, as well as in membrane-spanning helices. Here, we unravel the functional contribution of highly conserved transmembrane helix 1 (TM1) on the hASBT transport cycle. Consecutive cysteine substitution of individual residues along the TM1 helix (Ile 29 -Gly 50 ), as well as exofacial Asn 27 and Asn 28 , resulted in functional impairment of ϳ70% of mutants, despite appreciable cell surface expression for all but G50C. Cell surface expression of G50C and G50A was rescued upon MG132 treatment as well as cyclosporine A, but not by FK506 or bile acids, suggesting that Gly 50 is involved in hASBT folding. TM1 accessibility to membrane-impermeant MTSET remains confined to the exofacial half of the helix along a single, discrete face. Substrate protection from MTSET labeling was temperature-dependent for L34C, T36C, and L38C, consistent with conformational changes playing a role in solvent accessibility for these mutants. Residue Leu 30 was shown to be critical for both bile acid and sodium affinity, while Asn 27 , Leu 38 , Thr 39 , and Met 46 participate in sodium co-transport. Combined, our data demonstrate that TM1 plays a pivotal role in ASBT function and stability, thereby providing further insight in its dynamic transport mechanism.Enterohepatic recirculation is a highly efficient mechanism for conserving the body's total bile acid pool. Whereas the majority of bile acids are reabsorbed passively throughout the small intestine, active reabsorption occurs in the distal ileum by the apical sodium-dependent bile acid transporter (ASBT, 2 SLC10A2). As a high-capacity, high-affinity co-transporter, ASBT effectively reclaims the vast majority of bile acids, such that less than 5% of the circulating bile acid pool is lost through fecal elimination (1). Defective ASBT transport is associated with various disease conditions (2-4). Further, ASBT constitutes a pharmacologic target for improving oral drug bioavailability (5-7) as well as hypocholesterolemic agents, because cholesterol metabolism is induced upon bile acid depletion (8, 9). To elucidate the structure-function relationship of ASBT, our laboratory has previously employed cysteine scanning mutagenesis and site-directed alkylation techniques (10, 11) to determine structural requirements for substrates and their turnover (11-16). We demonstrate that residues lining TM6 (12) and TM7 (15) participate in substrate recognition and protein entry from the exofacial matrix, while the cytosolic half of TM3 mediates substrate release into the cytosolic milieu (14), putatively in conjunction with TM4 (18). Moreover, the extracellular loop (EL) 1 (13) and EL3 (16) regions mediate initial bile acid and sodium recognition and binding and may facili...

We report the involvement of transmembrane domain 4 (TM4) of hASBT in forming the putative translocation pathway, using cysteine-scanning mutagenesis in conjunction with solvent-accessibility studies using the membrane-impermeant, sulfhydryl-specific methanethiosulfonate reagents. We individually mutated each of the 21 amino acids in TM4 to cysteine on a fully functional, MTS-resistant C270A-hASBT template. The single-cysteine mutants were expressed in COS-1 cells, and their cell surface expression levels, transport activities [uptake of the prototypical hASBT substrate taurocholic acid (TCA)], and sensitivities to MTS exposure were determined. Only P161 lacked cell-surface expression. Overall, cysteine replacement was tolerated at charged and polar residues, except for mutants I160C, Y162C, I165C, and G179C (

CLC transporters catalyze the exchange of Cl- for H+ across cellular membranes. To do so, they must couple Cl- and H+ binding and unbinding to protein conformational change. However, the sole conformational changes distinguished crystallographically are small movements of a glutamate side chain that locally gates the ion-transport pathways. Therefore, our understanding of whether and how global protein dynamics contribute to the exchange mechanism has been severely limited. To overcome the limitations of crystallography, we used solution-state 13C-methyl NMR with labels on methionine, lysine, and engineered cysteine residues to investigate substrate (H+) dependent conformational change outside the restraints of crystallization. We show that methyl labels in several regions report H+-dependent spectral changes. We identify one of these regions as Helix R, a helix that extends from the center of the protein, where it forms the part of the inner gate to the Cl--permeation pathway, to the extracellular solution. The H+-dependent spectral change does not occur when a label is positioned just beyond Helix R, on the unstructured C-terminus of the protein. Together, the results suggest that H+ binding is mechanistically coupled to closing of the intracellular access-pathway for Cl-.

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