CXCR1 is one of two high-affinity receptors for the CXC chemokine interleukin-8 (IL-8), a major mediator of immune and inflammatory responses implicated in many disorders, including tumor growth1-3. IL-8, released in response to inflammatory stimuli, binds to the extracellular side of CXCR1. The ligand-activated intracellular signaling pathways result in neutrophil migration to the site of inflammation2. CXCR1 is a class-A, rhodopsin-like G-protein-coupled receptor (GPCR), the largest class of integral membrane proteins responsible for cellular signal transduction and targeted as drug receptors4-7. Despite its importance, its molecular mechanism is poorly understood due to the limited structural information available. Recently, structure determination of GPCRs has advanced by tailoring the receptors with stabilizing mutations, insertion of the protein T4 lysozyme and truncations of their amino acid sequences8, as well as addition of stabilizing antibodies and small molecules9 that facilitate crystallization in cubic phase monoolein mixtures10. The intracellular loops of GPCRs are critical for G-protein interactions11 and activation of CXCR1 involves both N-terminal residues and extracellular loops2,12,13. Our previous NMR studies indicate that IL-8 binding to the N-terminal residues is mediated by the membrane, underscoring the importance of the phospholipid bilayer for physiological activity14. Here we report the three-dimensional structure of human CXCR1 determined by NMR spectroscopy. The receptor is in liquid crystalline phospholipid bilayers, without modification of its amino acid sequence and under physiological conditions. Features important for intracellular G-protein activation and signal transduction are revealed.

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The ydgR gene of Escherichia coli encodes a protein of the proton-dependent oligopeptide transporter (POT) family. We cloned YdgR and overexpressed the His-tagged fusion protein in E. coli BL21 cells. Bacterial growth inhibition in the presence of the toxic phosphonopeptide alafosfalin established YgdR functionality. Transport was abolished in the presence of the proton ionophore carbonyl cyanide p-chlorophenylhydrazone, suggesting a proton-coupled transport mechanism. YdgR transports selectively only di-and tripeptides and structurally related peptidomimetics (such as aminocephalosporins) with a substrate recognition pattern almost identical to the mammalian peptide transporter PEPT1. The YdgR protein was purified to homogeneity from E. coli membranes. Blue native-polyacrylamide gel electrophoresis and transmission electron microscopy of detergent-solubilized YdgR suggest that it exists in monomeric form. Transmission electron microscopy revealed a crown-like structure with a diameter of ϳ8 nm and a central density. These are the first structural data obtained from a proton-dependent peptide transporter, and the YgdR protein seems an excellent model for studies on substrate and inhibitor interactions as well as on the molecular architecture of cell membrane peptide transporters.

CXCR1 is a receptor for the chemokine interleukin-8 (IL-8), a mediator of immune and inflammatory responses. Strategically located in the cell membrane, CXCR1 binds to IL-8 with high affinity, and subsequently transduces a signal across the membrane bilayer to a G-protein activated second messenger system. Here we describe NMR studies of the interactions between IL-8 and human CXCR1 in lipid environments. Functional full-length and truncated constructs of CXCR1 and full-length IL-8 were uniformly 15N-labeled by expression in bacteria followed by purification and refolding. The residues responsible for interactions between IL-8 and the N-terminal domain of CXCR1 were identified by specific chemical shift perturbations of assigned resonances on both IL-8 and CXCR1. Solution NMR signals from IL-8 in q=0.1 isotropic bicelles disappeared completely when CXCR1 in lipid bilayers was added in a 1:1 molar ratio, indicating that binding to the receptor-containing bilayers immobilizes IL-8 (on the ~105 Hz timescale) and broadens the signals beyond detection. The same solution NMR signals from IL-8 were less affected by the addition of N-terminal truncated CXCR1 in lipid bilayers, demonstrating that the N-terminal domain of CXCR1 is mainly responsible for binding to IL-8. The interaction is tight enough to immobilize IL-8 along with the receptor in phospholipid bilayers, and is specific enough to result in well-aligned samples in oriented sample solid-state NMR spectra. A combination of solution NMR and solid-state NMR studies of IL-8 in the presence of various constructs of CXCR1 enable us to propose a model for a multi-step binding process.

The local and global dynamics of the chemokine receptor CXCR1 are characterized using a combination of solution NMR and solid-state NMR experiments. In isotropic bicelles (q=0.1), only 13% of the expected number of backbone amide resonances is observed in 1H/15N HSQC solution NMR spectra of uniformly 15N labeled samples; extensive deuteration and the use of TROSY made little difference in the 800 MHz spectra. The limited number of observed amide signals are ascribed to mobile backbone sites, and assigned to specific residues in the protein; nineteen of the signals are from residues at the N-terminus and twenty-five from residues at the C-terminus. The solution NMR spectra display no evidence of local backbone motions from residues in the trans-membrane helices or inter-helical loops of CXCR1. This finding is reinforced by comparisons of solid-state NMR spectra of both magnetically aligned and unoriented bilayers containing either full-length or doubly N- and C-terminal truncated CXCR1 constructs. CXCR1 undergoes rapid rotational diffusion about the normal of liquid crystalline phospholipid bilayers; reductions in the frequency span and a change to axial symmetry are observed for both carbonyl carbon and amide nitrogen chemical shift powder patterns of unoriented samples containing 13C and 15N labeled CXCR1. In contrast, when the phospholipids are in the gel phase, CXCR1 does not undergo rapid global reorientation on the 104 Hz timescale defined by the carbonyl carbon and amide nitrogen chemical shift powder patterns.

The L-arginine/agmatine antiporter AdiC is a key component of the arginine-dependent extreme acid resistance system of Escherichia coli. Phylogenetic analysis indicated that AdiC belongs to the amino acid/polyamine/organocation (APC) transporter superfamily having sequence identities of 15-17% to eukaryotic and human APC transporters. For functional and structural characterization, we cloned, overexpressed, and purified wild-type AdiC and the point mutant AdiC-W293L, which is unable to bind and consequently transport L-arginine. Purified detergent-solubilized AdiC particles were dimeric. Reconstitution experiments yielded twodimensional crystals of AdiC-W293L diffracting beyond 6 Å resolution from which we determined the projection structure at 6.5 Å resolution. The projection map showed 10 -12 density peaks per monomer and suggested mainly tilted helices with the exception of one distinct perpendicular membrane spanning ␣-helix. Comparison of AdiC-W293L with the projection map of the oxalate/formate antiporter from Oxalobacter formigenes, a member from the major facilitator superfamily, indicated different structures. Thus, two-dimensional crystals of AdiC-W293L yielded the first detailed view of a transport protein from the APC superfamily at sub-nanometer resolution.Enteric pathogens such as Shigella, Salmonella, Yersinia, spp., and certain Escherichia coli strains can survive the extremely acidic conditions of the human stomach and cause intestinal diseases (1). To overcome the protective barrier of the gastric acidity, pathogenic and nonpathogenic strains of E. coli have developed acid resistance systems. One of these systems requires arginine to protect E. coli during low pH exposure. This arginine system is composed of an arginine-agmatine exchange transporter and of an acid-activated arginine decarboxylase (2). Acidification of the cytosol is prevented by the consumption of protons through decarboxylation of arginine to agmatine and carbon dioxide. Agmatine is then exported out of the cytosol, and new arginine is imported through the arginineagmatine transporter in a one-to-one exchange stoichiometry (2). This recently identified transport protein is the product of the adiC gene (3, 4). In vitro, AdiC-mediated exchange transport of arginine and agmatine is tightly coupled, electrogenic, and acid-activated (5). AdiC forms stable homodimers in detergent and phospholipid membranes as determined by gel filtration and glutaraldehyde cross-linking experiments (5).The origin of AdiC is somehow controversial as it was assigned to two families of transport proteins, i.e. the amino acid/polyamine/organocation (APC) 7 transporter superfamily (6) and the major facilitator superfamily (MFS) (5, 7). The APC superfamily of transporters consists of nearly 250 members that function as solute-cation symporters and solute-solute antiporters (6). According to hydropathy profile analysis and biochemically established topological features of most prokaryotic and eukaryotic APC superfamily members, both the N and C termini of ...

We have identified YkbA from Bacillus subtilis as a novel member of the L-amino acid transporter (LAT) family of amino acid transporters. The protein is ϳ30% identical in amino acid sequence to the light subunits of human heteromeric amino acid transporters. Purified His-tagged YkbA from Escherichia coli membranes reconstituted in proteoliposomes exhibited sodium-independent, obligatory exchange activity for L-serine and L-threonine and also for aromatic amino acids, albeit with less activity. Thus, we propose that YkbA be renamed SteT (Ser/Thr exchanger transporter). Kinetic analysis supports a sequential mechanism of exchange for SteT. Freeze-fracture analysis of purified, functionally active SteT in proteoliposomes, together with blue native polyacrylamide gel electrophoresis and transmission electron microscopy of detergent-solubilized purified SteT, suggest that the transporter exists in a monomeric form.Freeze-fracture analysis showed spherical particles with a diameter of 7.4 nm. Transmission electron microscopy revealed elliptical particles (diameters 6 ؋ 7 nm) with a distinct central depression. To our knowledge, this is the first functional characterization of a prokaryotic member of the LAT family and the first structural data on an APC (amino acids, polyamines, and choline for organocations) transporter. SteT represents an excellent model to study the molecular architecture of the light subunits of heteromeric amino acid transporters and other APC transporters.The APC (amino acids, polyamines, and choline for organocations) superfamily of transport proteins includes nearly 250 members that function as solute-cation symporters and solutesolute antiporters (1). They occur in all phyla from prokaryotes to higher eukaryotes and vary in length between 350 and 850 amino acid residues. The smaller proteins are generally of prokaryotic origin, whereas the larger ones are of eukaryotic origin and have N-and C-terminal hydrophilic extensions. Most APC members are predicted to possess 12 transmembrane (TM)The L-amino acid transporter (LAT) family belongs to the APC superfamily. LAT family members correspond to the light subunits of the heteromeric amino acid transporters (HATs), also called glycoprotein-associated amino acid transporters (2-4). HATs are composed of two subunits, a polytopic membrane protein (the light subunit) and a disulfide-linked N-glycosylated type II membrane glycoprotein (the heavy subunit). The light subunit is the catalytic component of the transporter, whereas the heavy subunit appears to be essential only for trafficking to the plasma membrane. Two types of heavy subunit (4F2hc and rBAT) and 10 types of light subunit have so far been *

Uptake of peptides into Escherichia coli cells is thought to be mediated by three different transport systems represented by the dipeptide permease Dpp, the tripeptide permease TppB, and the oligopeptide permease Opp [1][2][3]. Despite the fact that these proteins seem to discriminate according to the backbone length of peptide substrates, they do show some overlapping specificity. Although their prime physiological role is in the uptake of peptide-bound amino acids as an economic process to provide energy substrates and building blocks for cellular metabolism, peptide uptake seems also to be involved in signaling processes and metabolic adaptation [4]. Whereas Opp and Dpp represent ATP-binding cassette transporters with periplasmatic binding proteins [5][6][7], TppB belongs to the family of proton-dependent peptide symporters that utilize the proton gradient as driving force and lack cognate binding proteins [8]. After identification of the The genome of Escherichia coli contains four genes assigned to the peptide transporter (PTR) family. Of these, only tppB (ydgR) has been characterized, and named tripeptide permease, whereas protein functions encoded by the yhiP, ybgH and yjdL genes have remained unknown. Here we describe the overexpression of yhiP as a His-tagged fusion protein in E. coli and show saturable transport of glycyl-sarcosine (Gly-Sar) with an apparent affinity constant of 6.5 mm. Overexpression of the gene also increased the susceptibility of cells to the toxic dipeptide alafosfalin. Transport was strongly decreased in the presence of a protonophore but unaffected by sodium depletion, suggesting H + -dependence. This was confirmed by purification of YhiP and TppB by nickel affinity chromatography and reconstitution into liposomes. Both transporters showed Gly-Sar influx in the presence of an artificial proton gradient and generated transport currents on a chip-based sensor. Competition experiments established that YhiP transported dipeptides and tripeptides. Western blot analysis revealed an apparent mass of YhiP of 40 kDa. Taken together, these findings show that yhiP encodes a protein that mediates proton-dependent electrogenic transport of dipeptides and tripeptides with similarities to mammalian PEPT1. On the basis of our results, we propose to rename YhiP as DtpB (dipeptide and tripeptide permease B), by analogy with the nomenclature in other bacteria. We also propose to rename TppB as DtpA, to better describe its function as the first protein of the PTR family characterized in E. coli.Abbreviations AMCA, b-Ala-Lys-N