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
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...
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