Crystal structures of receptors involved in small molecule transport across membranes

This paper briefly reviews contemporary protein crystallography and focuses on six receptor proteins of membrane-intrinsic ATP binding cassette (ABC) transporters. Three of these receptors are specific for carbohydrates and three for amino acids. The receptor GacH of the transporter GacFGH from Streptomyces glaucescens is specific for acarbose and its homologs, and MalE of Salmonella typhimurium is specific for maltose but also forms a complex with acarbose, and the third receptor is the highly specific d-galactose receptor AcbH of the transporter AcbFGH from Actinoplanes sp. Concerning the receptors for amino acids, ArtJ belongs to the ArtJ-(MP)(2) transporter of Geobacillus stearotermophilus and recognizes and binds to positively charged arginine, lysine, and histidine with different sizes of side chains, contrasting the receptors Ngo0372 and Ngo2014 from Neisseria gonorrhaeae that are highly specific for cystine and cysteine, respectively. The differences in the rather unspecific receptors GacH, MalE and ArtJ are compared with the highly specific receptors AcbH, Ngo0372 and Ngo2014.     

A salt-bridge motif involved in ligand binding and large-scale domain motions of the maltose-binding protein

The uptake of nutrients is essential for the survival of bacterial cells. Many specialized systems have evolved, such as the maltose-dependent ABC transport system that transfers oligosaccharides through the cytoplasmic membrane. The maltose/maltodextrin-binding protein (MBP) serves as an initial high-affinity binding component in the periplasm that delivers the bound sugar into the cognate ABC transporter MalFGK(2). We have investigated the domain motions induced by the binding of the ligand maltotriose into the binding cleft using molecular dynamics simulations. We find that MBP is predominantly in the open state without ligand and in the closed state with ligand bound. Oligosaccharide binding induces a closure motion (30.0 degrees rotation), whereas ligand removal leads to domain opening (32.6 degrees rotation) around a well-defined hinge affecting key areas relevant for chemotaxis and transport. Our simulations suggest that a "hook-and-eye" motif is involved in the binding. A salt bridge between Glu-111 and Lys-15 forms that effectively locks the protein-ligand complex in a semiclosed conformation inhibiting any further opening and promoting complete closure. This previously unrecognized feature seems to secure the ligand in the binding site and keeps MBP in the closed conformation and suggests a role in the initial steps of substrate transport.     

Insights into the conformational equilibria of maltose-binding protein by analysis of high affinity mutants

The affinity of maltose-binding protein (MBP) for maltose and related carbohydrates was greatly increased by removal of groups in the interface opposite the ligand binding cleft. The wild-type protein has a KD of 1200 nM for maltose; mutation of residues Met-321 and Gln-325, both to alanine, resulted in a KD for maltose of 70 nM; deletion of 4 residues, Glu-172, Asn-173, Lys-175, and Tyr-176, which are part of a poorly ordered loop, results in a KD for maltose of 110 nM. Combining the mutations yields an increased affinity for maltodextrins and a KD of 6 nM for maltotriose. Comparison of ligand binding by the mutants, using surface plasmon resonance spectroscopy, indicates that decreases in the off-rate are responsible for the increased affinity. Small-angle x-ray scattering was used to demonstrate that the mutations do not significantly affect the solution conformation of MBP in either the presence or absence of maltose. The crystal structures of selected mutants showed that the mutations do not cause significant structural changes in either the closed or open conformation of MBP. These studies show that interactions in the interface opposite the ligand binding cleft, which we term the "balancing interface," are responsible for modulating the affinity of MBP for its ligand. Our results are consistent with a model in which the ligand-bound protein alternates between the closed and open conformations, and removal of interactions in the balancing interface decreases the stability of the open conformation, without affecting the closed conformation.     

The A-loop, a novel conserved aromatic acid subdomain upstream of the Walker A motif in ABC transporters, is critical for ATP binding

ATP-binding cassette (ABC) transporters represent one of the largest families of proteins, and transport a variety of substrates ranging from ions to amphipathic anticancer drugs. The functional unit of an ABC transporter is comprised of two transmembrane domains and two cytoplasmic ABC ATPase domains. The energy of the binding and hydrolysis of ATP is used to transport the substrates across membranes. An ABC domain consists of conserved regions, the Walker A and B motifs, the signature (or C) region and the D, H and Q loops. We recently described the A-loop (Aromatic residue interacting with the Adenine ring of ATP), a highly conserved aromatic residue approximately 25 amino acids upstream of the Walker A motif that is essential for ATP-binding. Here, we review the mutational analysis of this subdomain in human P-glycoprotein as well as homology modeling, structural and data mining studies that provide evidence for a functional role of the A-loop in ATP-binding in most members of the superfamily of ABC transporters.     

Uptake or extrusion: crystal structures of full ABC transporters suggest a common mechanism

ATP-binding cassette (ABC) transporters are integral membrane proteins that move diverse substrates across cellular membranes. ABC importers catalyse the uptake of essential nutrients from the environment, whereas ABC exporters facilitate the extrusion of various compounds, including drugs and antibiotics, from the cytoplasm. How ABC transporters couple ATP hydrolysis to the transport reaction has long remained unclear. The recent crystal structures of four complete ABC transporters suggest that a key step of the molecular mechanism is conserved in importers and exporters. Whereas binding of ATP promotes an outward-facing conformation, the release of the hydrolysis products ADP and phosphate promotes an inward-facing conformation. This basic scheme can in principle explain ATP-driven drug export and binding protein-dependent nutrient uptake.     

Structure and mechanism of ABC transporter proteins

ATP-binding cassette (ABC) transporters are ubiquitous membrane proteins that couple the transport of diverse substrates across cellular membranes to the hydrolysis of ATP. The crystal structures of four ABC transporters have recently been determined. They reveal similar arrangements of the conserved ATP-hydrolyzing nucleotide-binding domains, but unrelated architectures of the transmembrane domains, with the notable exception of a common 'coupling helix' that is essential for transmitting conformational changes. The structures suggest a mechanism that rationalizes ATP-driven transport: While binding of ATP appears to trigger an outward-facing conformation, dissociation of the hydrolysis products may promote an inward-facing conformation. This basic scheme can, in principle, explain nutrient import by ABC importers and drug extrusion by ABC exporters.     

Structure,Function, and Evolution of Bacterial ATP-Binding Cassette Systems

Summary: ATP-binding cassette (ABC) systems are universally distributed among living organisms and function in many different aspects of bacterial physiology. ABC transporters are best known for their role in the import of essential nutrients and the export of toxic molecules, but they can also mediate the transport of many other physiological substrates. In a classical transport reaction, two highly conserved ATP-binding domains or subunits couple the binding/hydrolysis of ATP to the translocation of particular substrates across the membrane, through interactions with membrane-spanning domains of the transporter. Variations on this basic theme involve soluble ABC ATP-binding proteins that couple ATP hydrolysis to nontransport processes, such as DNA repair and gene expression regulation. Insights into the structure, function, and mechanism of action of bacterial ABC proteins are reported, based on phylogenetic comparisons as well as classic biochemical and genetic approaches. The availability of an increasing number of high-resolution structures has provided a valuable framework for interpretation of recent studies, and realistic models have been proposed to explain how these fascinating molecular machines use complex dynamic processes to fulfill their numerous biological functions. These advances are also important for elucidating the mechanism of action of eukaryotic ABC proteins, because functional defects in many of them are responsible for severe human inherited diseases.     

Molecular Docking Simulations Provide Insights in the Substrate Binding Sites and Possible Substrates of the ABCC6 Transporter

The human ATP-binding cassette family C member 6 (ABCC6) gene encodes an ABC transporter protein (ABCC6), primarily expressed in liver and kidney. Mutations in the ABCC6 gene cause pseudoxanthoma elasticum (PXE), an autosomal recessive connective tissue disease characterized by ectopic mineralization of the elastic fibers. The pathophysiology underlying PXE is incompletely understood, which can at least partly be explained by the undetermined nature of the ABCC6 substrates as well as the unknown substrate recognition and binding sites. Several compounds, including anionic glutathione conjugates (N-ethylmaleimide; NEM-GS) and leukotriene C4 (LTC4) were shown to be modestly transported in vitro; conversely, vitamin K3 (VK3) was demonstrated not to be transported by ABCC6. To predict the possible substrate binding pockets of the ABCC6 transporter, we generated a 3D homology model of ABCC6 in both open and closed conformation, qualified for molecular docking and virtual screening approaches. By docking 10 reported in vitro substrates in our ABCC6 3D homology models, we were able to predict the substrate binding residues of ABCC6. Further, virtual screening of 4651 metabolites from the Human Serum Metabolome Database against our open conformation model disclosed possible substrates for ABCC6, which are mostly lipid and biliary secretion compounds, some of which are found to be involved in mineralization. Docking of these possible substrates in the closed conformation model also showed high affinity. Virtual screening expands this possibility to explore more compounds that can interact with ABCC6, and may aid in understanding the mechanisms leading to PXE.     

A specific interdomain interaction preserves the structural and binding properties of the ModA protein from the phytopathogen Xanthomonas citri domain interaction and transport in ModA

The periplasmic-binding proteins in ATP-binding cassette systems (ABC Transporters) are responsible for the capture and delivery of ligands to their specific transporters, triggering a series of ATP-driven conformational changes that leads to the transport of the ligand. Structurally consisting of two lobes, the proteins change conformation after interaction with the ligand. The structure of the molybdate-binding protein (ModA) from Xanthomonas citri, bound to molybdate, was previously solved by our group and an interdomain interaction, mediated by a salt bridge between K127 and D59, apparently supports the binding properties and keeps the domains closed. To determinate the importance of this interaction, we built two ModA mutants, K127S and D59A, and analysed their functional and structural properties. Based on a set of spectroscopic experiments, crystallisation trials, structure determination and molecular dynamics (MD) simulations, we showed that the salt bridge is essential to maintain the structure and binding properties. Additionally, the MD simulations revealed that this mutant adopted a more compact structure that packed down the ligand-binding pocket. From the closed bound to open structure, the positioning of the helices forming the dipole and the salt bridge are essential to induce an intermediate state.     

Evaluating the signal transduction mechanism of the parathyroid hormone 1 receptor. Effect of receptor-G-protein interaction on the ligand binding mechanism and receptor conformation

Ligand binding to the PTH1 receptor is described by a "two-site" model, in which the C-terminal portion of the ligand interacts with the N-terminal domain of the receptor (N interaction), and the N-terminal region of the ligand binds the juxtamembrane domain of the receptor (J interaction). Previous studies have not considered the dynamic nature of receptor conformation in ligand binding and receptor activation. In this study the ligand binding mechanism was compared for the G-protein-coupled (RG) and uncoupled (R) PTH1 receptor conformations. The two-site model was confirmed by demonstration of spatially distinct binding sites for PTH(3-34) and PTH(1-14): PTH(1-14), which binds predominantly to the J domain, only partially inhibited binding of 125I-PTH(3-34); and PTH(3-34), shown to bind predominantly to the N domain, only partially inhibited PTH(1-14)-stimulated cAMP accumulation. To assess the effect of R-G coupling, ligand binding to R was measured by displacement of 125I-PTH(3-34) with 30 microM guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) present, and binding to RG was measured by displacement of 125I-[MAP]PTHrP(1-36) (where MAP is model amphipathic peptide), a new radioligand that binds selectively to RG. Agonists bound with higher affinity to RG than R, whereas antagonists bound similarly to these states. The J interaction was responsible for enhanced agonist binding to RG: residues 1 and 2 were required for increased PTH(1-34) affinity for RG; residue 5 of MAP-PTHrP(1-36) was a determinant of R/RG binding selectivity, and PTH(1-14) bound selectively to RG. The N interaction was insensitive to R-G coupling; PTH(3-34) binding was GTPgammaS-insensitive. Finally, several observations suggest the receptor conformation is more "closed" at RG than R. At the R state, an open conformation is suggested by the simultaneous binding of PTH(1-14) and PTH(3-34). At RG PTH(1-14) better occluded binding of 125I-PTH(3-34) and agonist ligands bound pseudo-irreversibly, suggesting a more closed conformation of this receptor state. The results extend the two-site model to take into account R and RG conformations and suggest a model for differences of receptor conformation between these states.     

Alternating access to the transmembrane domain of the ATP-binding cassette protein cystic fibrosis transmembrane conductance regulator (ABCC7)

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a member of the ATP-binding cassette (ABC) protein family, most members of which act as active transporters. Actively transporting ABC proteins are thought to alternate between "outwardly facing" and "inwardly facing" conformations of the transmembrane substrate pathway. In CFTR, it is assumed that the outwardly facing conformation corresponds to the channel open state, based on homology with other ABC proteins. We have used patch clamp recording to quantify the rate of access of cysteine-reactive probes to cysteines introduced into two different transmembrane regions of CFTR from both the intracellular and extracellular solutions. Two probes, the large [2-sulfonatoethyl]methanethiosulfonate (MTSES) molecule and permeant Au(CN)(2)(-) ions, were applied to either side of the membrane to modify cysteines substituted for Leu-102 (first transmembrane region) and Thr-338 (sixth transmembrane region). Channel opening and closing were altered by mutations in the nucleotide binding domains of the channel. We find that, for both MTSES and Au(CN)(2)(-), access to these two cysteines from the cytoplasmic side is faster in open channels, whereas access to these same sites from the extracellular side is faster in closed channels. These results are consistent with alternating access to the transmembrane regions, however with the open state facing inwardly and the closed state facing outwardly. Our findings therefore prompt revision of current CFTR structural and mechanistic models, as well as having broader implications for transport mechanisms in all ABC proteins. Our results also suggest possible locations of both functional and dysfunctional ("vestigial") gates within the CFTR permeation pathway.     

Binding of glutamate to the umami receptor

The umami taste receptor is a heterodimer composed of two members of the T1R taste receptor family: T1R1 and T1R3. It detects glutamate in humans, and is a more general amino acid detector in other species. We have constructed homology models of the ligand binding domains of the human umami receptor (based on crystallographic structures of the metabotropic glutamate receptor of the central nervous system). We have carried out molecular dynamics simulations of the ligand binding domains, and we find that the likely conformation is that T1R1 receptor protein exists in the closed conformation, and T1R3 receptor in the open conformation in the heterodimer. Further, we have identified the important binding interactions and have made an estimate of the relative free energies associated with the two glutamate binding sites.     

Crystal structures of the choline/acetylcholine substrate-binding protein ChoX from Sinorhizobium meliloti in the liganded and unliganded-closed states

The ATP-binding cassette transporter ChoVWX is one of several choline import systems operating in Sinorhizobium meliloti. Here fluorescence-based ligand binding assays were used to quantitate substrate binding by the periplasmic ligand-binding protein ChoX. These data confirmed that ChoX recognizes choline and acetylcholine with high and medium affinity, respectively. We also report the crystal structures of ChoX in complex with either choline or acetylcholine. These structural investigations revealed an architecture of the ChoX binding pocket and mode of substrate binding similar to that reported previously for several compatible solute-binding proteins. Additionally the ChoX-acetylcholine complex permitted a detailed structural comparison with the carbamylcholine-binding site of the acetylcholine-binding protein from the mollusc Lymnaea stagnalis. In addition to the two liganded structures of ChoX, we were also able to solve the crystal structure of ChoX in a closed, substrate-free conformation that revealed an architecture of the ligand-binding site that is superimposable to the closed, ligand-bound form of ChoX. This structure is only the second of its kind and raises the important question of how ATP-binding cassette transporters are capable of distinguishing liganded and unliganded-closed states of the binding protein.     

Structure of nucleotide-binding domain 1 of the cystic fibrosis transmembrane conductance regulator

Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a chloride channel. Nucleotide-binding domain 1 (NBD1), one of two ABC domains in CFTR, also contains sites for the predominant CF-causing mutation and, potentially, for regulatory phosphorylation. We have determined crystal structures for mouse NBD1 in unliganded, ADP- and ATP-bound states, with and without phosphorylation. This NBD1 differs from typical ABC domains in having added regulatory segments, a foreshortened subdomain interconnection, and an unusual nucleotide conformation. Moreover, isolated NBD1 has undetectable ATPase activity and its structure is essentially the same independent of ligand state. Phe508, which is commonly deleted in CF, is exposed at a putative NBD1-transmembrane interface. Our results are consistent with a CFTR mechanism, whereby channel gating occurs through ATP binding in an NBD1-NBD2 nucleotide sandwich that forms upon displacement of NBD1 regulatory segments.     

The extracellular domains of ErbB3 retain high ligand binding affinity at endosome pH and in the locked conformation

The extracellular, ligand binding regions of ErbB receptors consist of four domains that can assume at least two alternative conformations, extended and locked. The locked conformation, observed in several crystal structures, is held together by a noncovalent intramolecular tether and is incompatible with current models for receptor dimerization and ligand activation. Based on structures of ligand-receptor complexes in the extended conformation, the high affinity ligand binding pocket between domains I and III is disrupted in the locked conformation. Therefore the biological role of the locked conformation is not clear. To address the impact of the locked conformation on ligand binding, we compared extracellular domains of wild-type ErbB3, mutant domains in a constitutively locked or extended conformation and partial extracellular domain constructs. We found that the constitutively locked receptor domains and truncated constructs carrying only domains I-II or III-IV strongly bind ligand, albeit with reduced affinity compared to wild-type receptor. This suggests that the locked conformation cannot be discounted for ligand binding. The significant binding by both partial interfaces in domains I and III also suggests that "partial bivalency" may be the reason for the low nanomolar and high picomolar binding observed for ErbB3 in the respective "low" and high affinity states. In contrast to EGFR (ErbB1), ErbB3 retains high ligand binding affinity at an endosome-comparable pH in both the extended and locked conformations. Ligand affinity for the locked conformation even improves at low pH. For ErbB3, the contribution of domain I to ligand binding is strong and increases at low pH while its contribution is thought to be minimal for EGFR, regardless of pH. This shift in domain contribution and pH dependency provides a mechanistic explanation for some of the divergent properties of EGFR and ErbB3.     

Resistance of Neisseria gonorrhoeae to non-oxidative killing by adherent human polymorphonuclear leucocytes

Symptomatic infection with Neisseria gonorrhoeae (Gc) is characterized by abundant neutrophil (PMN, polymorphonuclear leucocyte) influx, but PMNs cannot clear initial infection, indicating that Gc possess defences against PMN challenge. In this study, survival of liquid-grown Gc was monitored after synchronous infection of adherent, interleukin 8-treated human PMNs. 40–70% of FA1090 Gc survived 1 h of PMN exposure, after which bacterial numbers increased. Assays with bacterial viability dyes along with soybean lectin to detect extracellular Gc revealed that a subset of both intracellular and extracellular PMN-associated Gc were viable. Gc survival was unaffected in PMNs chemically or genetically deficient for producing reactive oxygen species (ROS). This result held true even for OpaB+ Gc, which stimulate neutrophil ROS production. Catalase- and RecA-deficient Gc, which are more sensitive to ROS in vitro , had no PMN survival defect. recN and ngo1686 mutant Gc also exhibit increased sensitivity to ROS and PMNs, but survival of these mutants was not rescued in ROS-deficient cells. The ngo1686 mutant showed increased sensitivity to extracellular but not intracellular PMN killing. We conclude that Gc are remarkably resistant to PMN killing, killing occurs independently of neutrophil ROS production and Ngo1686 and RecN defend Gc from non-oxidative PMN antimicrobial factors.     

Molecular Dynamics Simulation of the Allosteric Regulation of eIF4A Protein from the Open to Closed State,Induced by ATP and RNA Substrates

Background

Eukaryotic initiation factor 4A (eIF4A) plays a key role in the process of protein translation initiation by facilitating the melting of the 5′ proximal secondary structure of eukaryotic mRNA for ribosomal subunit attachment. It was experimentally postulated that the closed conformation of the eIF4A protein bound by the ATP and RNA substrates is coupled to RNA duplex unwinding to promote protein translation initiation, rather than an open conformation in the absence of ATP and RNA substrates. However, the allosteric process of eIF4A from the open to closed state induced by the ATP and RNA substrates are not yet fully understood.

Methodology

In the present work, we constructed a series of diplex and ternary models of the eIF4A protein bound by the ATP and RNA substrates to carry out molecular dynamics simulations, free energy calculations and conformation analysis and explore the allosteric properties of eIF4A.

Results

The results showed that the eIF4A protein completes the conformational transition from the open to closed state via two allosteric processes of ATP binding followed by RNA and vice versa. Based on cooperative allosteric network analysis, the ATP binding to the eIF4A protein mainly caused the relative rotation of two domains, while the RNA binding caused the proximity of two domains via the migration of RNA bases in the presence of ATP. The cooperative binding of ATP and RNA for the eIF4A protein plays a key role in the allosteric transition.     

Syndecan-1 and syndecan-4 can mediate the invasion of OpaHSPG-expressing Neisseria gonorrhoeae into epithelial cells

Neisseria gonorrhoeae ( Ngo ) expressing the outer membrane protein Opa_HSPG can adhere to and invade epithelial cells via binding to heparan sulphate proteoglycan (HSPG) receptors. In this study, we have investigated the role of syndecan-1 and syndecan-4, two members of the HSPG family, in the uptake of Ngo by epithelial cells. When overexpressed in HeLa cells, both syndecans co-localize with adherent Ngo on the host cell surface. This overexpression of syndecan-1 and syndecan-4 leads to a three- and sevenfold increase in Ngo invasion respectively. In contrast, transfection with the syndecan-1 and syndecan-4 mutant constructs lacking the intracellular domain results in an abrogation of the invasion process, characteristic of a dominant-negative mode of action. A concomitant loss of the capacity to mediate Ngo uptake was also observed with syndecan-4 mutant constructs carrying lesions in the dimerization motif necessary for the binding of protein kinase C (PKC) and phosphatidylinositol 4,5-bisphosphate (PIP₂), and mutants that are deficient in a C-terminal EFYA amino acid motif responsible for binding to syntenin or CASK. We conclude that syndecan-1 and syndecan-4 can both mediate Ngo uptake into epithelial cells, and that their intracellular domains play a crucial role in this process, perhaps by mediating signal transduction or anchorage to the cytoskeleton.     

Transmembrane gate movements in the type II ATP-binding cassette (ABC) importer BtuCD-F during nucleotide cycle

ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins that translocate substrates across cell membranes. The alternating access of their transmembrane domains to opposite sides of the membrane powered by the closure and reopening of the nucleotide binding domains is proposed to drive the translocation events. Despite clear structural similarities, evidence for considerable mechanistic diversity starts to accumulate within the importers subfamily. We present here a detailed study of the gating mechanism of a type II ABC importer, the BtuCD-F vitamin B(12) importer from Escherichia coli, elucidated by EPR spectroscopy. Distance changes at key positions in the translocation gates in the nucleotide-free, ATP- and ADP-bound conformations of the transporter were measured in detergent micelles and liposomes. The translocation gates of the BtuCD-F complex undergo conformational changes in line with a "two-state" alternating access model. We provide the first direct evidence that binding of ATP drives the gates to an inward-facing conformation, in contrast to type I importers specific for maltose, molybdate, or methionine. Following ATP hydrolysis, the translocation gates restore to an apo-like conformation. In the presence of ATP, an excess of vitamin B(12) promotes the reopening of the gates toward the periplasm and the dislodgment of BtuF from the transporter. The EPR data allow a productive translocation cycle of the vitamin B(12) transporter to be modeled.     

Membrane reconstitution of ABC transporters and assays of translocator function

In this protocol, we describe a procedure for incorporating ATP-binding cassette (ABC) transporters into large unilamellar vesicles (LUVs) and assays to determine ligand binding and solute translocation by these membrane-reconstituted systems. The reconstitution technique as described has been optimized for ABC transporters but can be readily adapted for other types of transport systems. Purified transporters are inserted into detergent-destabilized preformed liposomes and detergent is subsequently removed by adsorption onto polystyrene beads. Next, Mg-ATP or an ATP-regenerating system is incorporated into the vesicle lumen by one or more cycles of freezing-thawing, followed by extrusion through polycarbonate filters to obtain unilamellar vesicles. Binding and translocation of substrates are measured using isotope-labeled ligands and rapid filtration to separate the proteoliposomes from the surrounding medium. Quantitative information is obtained about dissociation constants (K(d)) for ligand binding, number of binding-sites, transport affinities (K(m)), rates of transport, and the activities of transporter molecules with opposite orientations in the membrane. The full protocol can be completed within 4-5 d.