Klaas M. Pos

Basolateral aromatic amino acid transporter TAT1 (Slc16a10) functions as an efflux pathway

Basolateral efflux is a necessary step in transepithelial (re)absorption of amino acids from small intestine and kidney proximal tubule. The best characterized basolateral amino acid transporters are y+LAT1‐4F2hc and LAT2‐4F2hc that function as obligatory exchangers and thus, do not contribute to net amino acid (re)absorption. The aromatic amino acid transporter TAT1 was shown previously to localize basolaterally in rats small intestine and to mediate the efflux of L‐Trp in the absence of exchange substrate, upon expression in Xenopus oocytes. We compared here the amino acid influx and efflux via mouse TAT1 in Xenopus oocytes. The results show that mTAT1 functions as facilitated diffusion pathway for aromatic amino acids and that its properties are symmetrical in terms of selectivity and apparent affinity. We show by real‐time RT‐PCR that its mRNA is highly expressed in mouse small intestine mucosa, kidney, liver, and skeletal muscle as well as present in all other tested tissues. We show that mTAT1 is not N‐glycosylated and that it localizes to the mouse kidney proximal tubule. This expression is characterized by an axial gradient similar to that of the luminal neutral amino acid transporter B0AT1 and shows the same basolateral localization as 4F2hc. mTAT1 also localizes to the basolateral membrane of small intestine enterocytes and to the sinusoidal side of perivenous hepatocytes. In summary, we show that TAT1 is a basolateral epithelial transporter and that it can function as a net efflux pathway for aromatic amino acids. We propose that it, thereby, may supply parallel exchangers with recycling uptake substrates that could drive the efflux of other amino acids. J.Cell.Physiol.

Tripartite assembly of RND multidrug efflux pumps.

Tripartite multidrug efflux systems of Gram-negative bacteria are composed of an inner membrane transporter, an outer membrane channel and a periplasmic adaptor protein. They are assumed to form ducts inside the periplasm facilitating drug exit across the outer membrane. Here we present the reconstitution of native Pseudomonas aeruginosa MexAB–OprM and Escherichia coli AcrAB–TolC tripartite Resistance Nodulation and cell Division (RND) efflux systems in a lipid nanodisc system. Single-particle analysis by electron microscopy reveals the inner and outer membrane protein components linked together via the periplasmic adaptor protein. This intrinsic ability of the native components to self-assemble also leads to the formation of a stable interspecies AcrA–MexB–TolC complex suggesting a common mechanism of tripartite assembly. Projection structures of all three complexes emphasize the role of the periplasmic adaptor protein as part of the exit duct with no physical interaction between the inner and outer membrane components.

Molecular basis for inhibition of AcrB multidrug efflux pump by novel and powerful pyranopyridine derivatives

Significance AcrB is one of the major multidrug resistance-conferring antibiotic efflux pumps from pathogenic bacteria. We have designed and produced the periplasmic, substrate binding domain of AcrB and solved its crystal structure in complex with multiple novel pyranopyridine inhibitors, as well as with drugs transported by AcrB. The structural data are corroborated by various cellular assays and molecular dynamics (MD) simulations, and allow us to propose a mechanism for AcrB efflux inhibition. Furthermore, the results provide a molecular platform for the development of combinational therapies against pathogenic Enterobacteriaceae. The Escherichia coli AcrAB-TolC efflux pump is the archetype of the resistance nodulation cell division (RND) exporters from Gram-negative bacteria. Overexpression of RND-type efflux pumps is a major factor in multidrug resistance (MDR), which makes these pumps important antibacterial drug discovery targets. We have recently developed novel pyranopyridine-based inhibitors of AcrB, which are orders of magnitude more powerful than the previously known inhibitors. However, further development of such inhibitors has been hindered by the lack of structural information for rational drug design. Although only the soluble, periplasmic part of AcrB binds and exports the ligands, the presence of the membrane-embedded domain in AcrB and its polyspecific binding behavior have made cocrystallization with drugs challenging. To overcome this obstacle, we have engineered and produced a soluble version of AcrB [AcrB periplasmic domain (AcrBper)], which is highly congruent in structure with the periplasmic part of the full-length protein, and is capable of binding substrates and potent inhibitors. Here, we describe the molecular basis for pyranopyridine-based inhibition of AcrB using a combination of cellular, X-ray crystallographic, and molecular dynamics (MD) simulations studies. The pyranopyridines bind within a phenylalanine-rich cage that branches from the deep binding pocket of AcrB, where they form extensive hydrophobic interactions. Moreover, the increasing potency of improved inhibitors correlates with the formation of a delicate protein- and water-mediated hydrogen bond network. These detailed insights provide a molecular platform for the development of novel combinational therapies using efflux pump inhibitors for combating multidrug resistant Gram-negative pathogens.

An antibody library for stabilizing and crystallizing membrane proteins - selecting binders to the citrate carrier CitS

Co‐crystallization of membrane proteins with antibody fragments may emerge as a general tool to facilitate crystal growth and improve crystal quality. The bound antibody fragment enlarges the hydrophilic part of the mostly hydrophobic membrane protein, thereby increasing the interaction area for possible protein–protein contacts in the crystal. Additionally, it may restrain flexible parts or lock the membrane protein in a defined conformational state. For successful co‐crystallization trials, the antibody fragments must be stable in detergents during the extended period of crystal growth and must be easily produced in amounts necessary for crystallography. Therefore, we constructed a library of antibody Fab fragments from a framework subset of the HuCAL GOLD® library (Morphosys, Munich, Germany). By combining the most stable and well expressed frameworks, VH3 and Vκ3, with the further stabilizing constant domains, a Fab library with the desired properties was obtained in a standard phage display format. As a proof of principle, we selected binders with phage display against the detergent‐solubilized citrate transporter CitS of Klebsiella pneumoniae. We describe efficient methods for the immobilization of the membrane protein during selection, for ELISA screening, and for BIAcore evaluation. We demonstrate that the selected Fab fragments form stable complexes with native CitS and recognize conformational epitopes with affinities in the low nanomolar range.

Recycling of aromatic amino acids via TAT1 allows efflux of neutral amino acids via LAT2-4F2hc exchanger

The rate of amino acid efflux from individual cells needs to be adapted to cellular demands and plays a central role for the control of extracellular amino acid homeostasis. A particular example of such an outward amino acid transport is the basolateral efflux from transporting epithelial cells located in the small intestine and kidney proximal tubule. Because LAT2-4F2hc (Slc7a8–Slc3a2), the best known basolateral neutral amino acid transporter of these epithelial cells, functions as an obligatory exchanger, we tested whether TAT1 (Slc16a10), the aromatic amino-acid facilitated diffusion transporter, might allow amino acid efflux via this exchanger by recycling its influx substrates. In this study, we show by immunofluorescence that TAT1 and LAT2 indeed colocalize in the early kidney proximal tubule. Using the Xenopus laevis oocytes expression system, we show that l-glutamine is released from oocytes into an amino-acid-free medium only when both transporters are coexpressed. High-performance liquid chromatography analysis reveals that several other neutral amino acids are released as well. The transport function of both TAT1 and LAT2-4F2hc is necessary for this efflux, as coexpression of functionally inactive but surface-expressed mutants is ineffective. Based on negative results of coimmunoprecipitation and crosslinking experiments, the physical interaction of these transporters does not appear to be required. Furthermore, replacement of TAT1 or LAT2-4F2hc by the facilitated diffusion transporter LAT4 or the obligatory exchanger LAT1, respectively, supports similar functional cooperation. Taken together, the results suggest that the aromatic amino acid diffusion pathway TAT1 can control neutral amino acid efflux via neighboring exchanger LAT2-4F2hc, by recycling its aromatic influx substrates.

Crystallographic analysis of AcrB

A His‐tagged derivative of the multidrug efflux pump AcrB could be crystallized in three different space groups (R3, R32 and P321). Experimental MAD‐phasing maps from R32 AcrBHis crystals were obtained to a resolution of 3.5 Å. Datasets of native and substrate soaked AcrBHis crystals were collected at the Swiss Light Source X06SA beamline up to a resolution of 2.7 Å and refinement of these data provided good quality electron density maps, which allowed us to complement the published AcrB structure (PDB code 1iwg). Introduction of amino acids 860–865 and 868 lacking in the 1iwg structure and deletion of a highly disordered region (amino acids 669–678) improved R free and average B factors in the 2.7 Å model. We could not identify significant densities indicating specific antibiotic binding sites in the AcrB R32 space group datasets under the soaking conditions tested.

Purification, crystallization and preliminary diffraction studies of AcrB, an inner-membrane multi-drug efflux protein.

Resistance of pathogens to antibiotics is often dependent on multi-drug export proteins that reside in the inner membrane of bacteria. This work describes the expression, purification, crystallization and preliminary crystallographic analysis of AcrB of Escherichia coli. Together with AcrA and TolC, AcrB forms a proton motive force dependent efflux pump of the resistance-nodulation-cell division (RND) transporter superfamily and is responsible for resistance towards many common antibiotics such as ciprofloxacin and novobiocin. AcrB crystallizes in space group R32, with unit-cell parameters a = b = 143, c = 513 A; the crystals diffract to 3.0 A resolution.

Mechanisms of envelope permeability and antibiotic influx and efflux in Gram-negative bacteria

Researchers, clinicians and governments all recognize antimicrobial resistance as a serious and growing threat worldwide. New antimicrobials are urgently needed, especially for infections caused by Gram-negative bacteria, whose cell envelopes are characterized by low permeability and often contain drug efflux systems. Individual bacteria and populations control their internal concentrations of antibiotics by regulating proteins involved in membrane permeability, such as porins or efflux pumps. Robust methods to quantify and visualize intrabacterial antibiotic concentrations have identified clear correlations between efflux activity and drug diffusion and accumulation in both susceptible and resistant strains, and have also clarified how certain chemical structures can affect drug entry and residence time within the cell. In this PERSPECTIVE, we discuss the biological underpinnings of drug permeability and export using several prototypical influx and efflux systems. We also highlight how new methods for the determination of antibacterial activities enable more careful quantitation and may provide us with a way forward for capturing and correlating the modes of action and kinetics of antibiotic uptake inside bacterial cells. Together, these advances will aid efforts to generate structurally improved molecules with better access and retention within bacteria, thereby reducing the emergence and spread of resistant strains and extending the clinical use of current antibiotics. A Perspective on unravelling the mechanisms of antibiotic penetration and efflux in Gram-negative bacteria.

Characterization of the citrate/acetate antiporter CitW of Klebsiella pneumoniae.

Abstract. The genome of Klebsiella pneumoniae contains at least three different genes encoding citrate transporters. Recently, a third and hitherto unknown gene encoding a citrate transport system (citW) was identified. Escherichia coli transformed with a plasmid expressing citW was able to grow on citrate as sole carbon and energy source, identifying CitW as a citrate carrier. In this report, we provide evidence that further specifies CitW as a Na+-independent citrate/citrate and citrate/acetate exchanger. Kinetic analysis of citrate uptake at different pH values identified Hcitrate2– as the transported citrate species, with a Km of 25xa0µM. Since citW is expressed under anoxic conditions and acetate is the main end-product of citrate fermentation in K. pneumoniae, citrate/acetate exchange might be its in vivo function. Sequence similarity searches identified CitW (454 amino acids, 48.15xa0kDa) as a member of the 2-hydroxycarboxylate transporter family (TC 2.A.24). The substrate specificity seems to partially contradict this phylogenetic classification, but appears logical with respect to the putative functional role of CitW in the citrate fermentation pathway of K. pneumoniae.

Crystal structure and mechanistic basis of a functional homolog of the antigen transporter TAP

Significance ABC transporters shuttle chemically diverse substances across membranes in an energy-dependent manner. They mediate multidrug resistance in microorganisms and cancer cells and can cause human pathologies when dysfunctional. Although important insights into ABC transporters have been gained in recent years, fundamental questions concerning their mechanism remain open. Here, we identify the protein complex TmrAB as a functional homolog of the antigenic peptide transporter TAP and present its high-resolution structure. The structure adopts an asymmetric conformational state and is characterized by C-terminal zipper helices that are essential for efficient substrate translocation. The structure, together with functional studies, enables us to outline the general conformational dynamics of heterodimeric ABC transporters and to establish TmrAB as a model system for TAP. ABC transporters form one of the largest protein superfamilies in all domains of life, catalyzing the movement of diverse substrates across membranes. In this key position, ABC transporters can mediate multidrug resistance in cancer therapy and their dysfunction is linked to various diseases. Here, we describe the 2.7-Å X-ray structure of heterodimeric Thermus thermophilus multidrug resistance proteins A and B (TmrAB), which not only shares structural homology with the antigen translocation complex TAP, but is also able to restore antigen processing in human TAP-deficient cells. TmrAB exhibits a broad peptide specificity and can concentrate substrates several thousandfold, using only one single active ATP-binding site. In our structure, TmrAB adopts an asymmetric inward-facing state, and we show that the C-terminal helices, arranged in a zipper-like fashion, play a crucial role in guiding the conformational changes associated with substrate transport. In conclusion, TmrAB can be regarded as a model system for asymmetric ABC exporters in general, and for TAP in particular.