Revealing an outward-facing open conformational state in a CLC Cl(-)/H(+) exchange transporter.

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 (19)F 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.

13C NMR detects conformational change in the 100-kD membrane transporter ClC-ec1.

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 (13)C-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(-).

The Structure and Function of OxlT, the Oxalate Transporter of Oxalobacter formigenes.

OxlT, the oxalate transporter of Oxalobacter formigenes, is a member of the Major Facilitator Superfamily of transporters (MFS), one of the largest groups of membrane proteins with substantial relevance to solute transport physiology, pharmacology, and possible drug development. MFS proteins transport a wide range of substrates such as organic and inorganic anions, sugars, drugs, and neurotransmitters. This review succinctly summarizes experimental work on a model MFS protein, OxlT, beginning with its identification as an electrogenic oxalate/formate exchanger, its three-dimensional structure, and discussion of biochemical and biophysical data that have shed further light on its structure and function. We also discuss the structure and function of OxlT in relation to notable MFS carriers such as LacY and GlpT.

Transmembrane helix 1 contributes to substrate translocation and protein stability of bile acid transporter SLC10A2.

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 ∼70% 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.

Bacterial peptide recognition and immune activation facilitated by human peptide transporter PEPT2.

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 gamma-D-glutamyl-meso-diaminopimelic acid (gamma-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 gamma-iE-DAP uptake was mediated by PEPT2 with an affinity constant of approximately 193 microM, whereas muramyl dipeptide was not transported. Exposure to gamma-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.

Cytosolic half of transmembrane domain IV of the human bile acid transporter hASBT (SLC10A2) forms part of the substrate translocation pathway.

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 (

Design of novel synthetic MTS conjugates of bile acids for site-directed sulfhydryl labeling of cysteine residues in bile acid binding and transporting proteins.

The purpose of this study was to design bile acid-containing methanethiosulfonate (MTS) agents with appropriate physical attributes to effectively modify the cysteine residues present in the human apical sodium-dependent bile acid transporter. Four physical properties including surface area, molecular volume, ClogP, and dipole moment were calculated for each semiempirically optimized structure of MTS compounds. The specificity of the synthesized bile acid-MTS conjugate toward native cysteines and putative bile acid interacting domains of hASBT was supported by the effect of 1mM cholyl-MTS, cholylglycyl-MTS, and 3-amino-cholyl-MTS on uptake activity, that displayed a significant decrease in TCA affinity (K(T)=69.9+/-4.5, 69.01+/-6.2, and 63.24+/-0.26 microM and J(max)=35.8+/-0.3, 24.03+/-1.22, 46.49+/-5.01 pmol mg protein min(-1), respectively). These compounds prove to be effective tools in probing the structural and functional effects of cysteine residues in bile acid binding and transporting proteins.

Topology scanning and putative three-dimensional structure of the extracellular binding domains of the apical sodium-dependent bile acid transporter (SLC10A2).

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.

Current perspectives on the cellular uptake and trafficking of riboflavin.

The role of riboflavin in cell maintenance and growth, and the mechanism by which it is absorbed into various human tissues and cell lines has been extensively studied over the past decade. Evidence suggests two absorption mechanisms, a saturable-active component that dominates at near physiological vitamin concentrations and a passive component that is revealed at oversupplemented riboflavin conditions. Various transport modulator studies consistently suggest a highly riboflavin specific, temperature-dependent active transport mechanism that is regulated by the Ca2+/calmodulin pathway. The PKA and PKG pathways have also been implicated in absorption regulation. The long-standing model that riboflavin absorption involves a carrier-mediated transporter has recently been challenged through studies suggesting a receptor-mediated endocytic component. The presence of a soluble, human riboflavin binding protein in the transport stratagem has been shown to play an important role in fetal development. The relationship of this binding protein with the riboflavin specific membrane bound protein, though currently not well defined, may involve a protein-protein interaction that plays a primary role in this proposed receptor-mediated component.