Structural and mechanistic analysis of a tripartite ATP-independent periplasmic TRAP transporter.

Tripartite ATP-independent periplasmic (TRAP) transporters are found widely in bacteria and archaea and consist of three structural domains, a soluble substrate-binding protein (P-domain), and two transmembrane domains (Q- and M-domains). HiSiaPQM and its homologs are TRAP transporters for sialic acid and are essential for host colonization by pathogenic bacteria. Here, we reconstitute HiSiaQM into lipid nanodiscs and use cryo-EM to reveal the structure of a TRAP transporter. It is composed of 16 transmembrane helices that are unexpectedly structurally related to multimeric elevator-type transporters. The idiosyncratic Q-domain of TRAP transporters enables the formation of a monomeric elevator architecture. A model of the tripartite PQM complex is experimentally validated and reveals the coupling of the substrate-binding protein to the transporter domains. We use single-molecule total internal reflection fluorescence (TIRF) microscopy in solid-supported lipid bilayers and surface plasmon resonance to study the formation of the tripartite complex and to investigate the impact of interface mutants. Furthermore, we characterize high-affinity single variable domains on heavy chain (VHH) antibodies that bind to the periplasmic side of HiSiaQM and inhibit sialic acid uptake, providing insight into how TRAP transporter function might be inhibited in vivo.

Hyperosmotic Stress Allosterically Reconfigures Betaine Binding Pocket in BetP.

The transporter BetP in C. glutamicum is essential in maintaining bacterial cell viability during hyperosmotic stress and functions by co-transporting betaine and Na+ into bacterial cells. Hyperosmotic stress leads to increased intracellular K+ concentrations which in turn promotes betaine binding. While structural details of multiple end state conformations of BetP have provided high resolution snapshots, how K+ sensing by the C-terminal domain is allosterically relayed to the betaine binding site is not well understood. In this study, we describe conformational dynamics in solution of BetP using amide hydrogen/deuterium exchange mass spectrometry. These reveal how K+ alters conformation of the disordered C- and N-terminal domains to allosterically reconfigure transmembrane helices 3, 8, and 10 to enhance betaine interactions. A map of the betaine binding site, at near single amino acid resolution, reveals a critical extrahelical H-bond mediated by TM3 with betaine.

The Vibrio vulnificus stressosome is an oxygen-sensor involved in regulating iron metabolism.

Stressosomes are stress-sensing protein complexes widely conserved among bacteria. Although a role in the regulation of the general stress response is well documented in Gram-positive bacteria, the activating signals are still unclear, and little is known about the physiological function of stressosomes in the Gram-negative bacteria. Here we investigated the stressosome of the Gram-negative marine pathogen Vibrio vulnificus. We demonstrate that it senses oxygen and identified its role in modulating iron-metabolism. We determined a cryo-electron microscopy structure of the VvRsbR:VvRsbS stressosome complex, the first solved from a Gram-negative bacterium. The structure points to a variation in the VvRsbR and VvRsbS stoichiometry and a symmetry breach in the oxygen sensing domain of VvRsbR, suggesting how signal-sensing elicits a stress response. The findings provide a link between ligand-dependent signaling and an output - regulation of iron metabolism - for a stressosome complex.

Extracellular citrate and metabolic adaptations of cancer cells.

It is well established that cancer cells acquire energy via the Warburg effect and oxidative phosphorylation. Citrate is considered to play a crucial role in cancer metabolism by virtue of its production in the reverse Krebs cycle from glutamine. Here, we review the evidence that extracellular citrate is one of the key metabolites of the metabolic pathways present in cancer cells. We review the different mechanisms by which pathways involved in keeping redox balance respond to the need of intracellular citrate synthesis under different extracellular metabolic conditions. In this context, we further discuss the hypothesis that extracellular citrate plays a role in switching between oxidative phosphorylation and the Warburg effect while citrate uptake enhances metastatic activities and therapy resistance. We also present the possibility that organs rich in citrate such as the liver, brain and bones might form a perfect niche for the secondary tumour growth and improve survival of colonising cancer cells. Consistently, metabolic support provided by cancer-associated and senescent cells is also discussed. Finally, we highlight evidence on the role of citrate on immune cells and its potential to modulate the biological functions of pro- and anti-tumour immune cells in the tumour microenvironment. Collectively, we review intriguing evidence supporting the potential role of extracellular citrate in the regulation of the overall cancer metabolism and metastatic activity.

A polycystin-2 protein with modified channel properties leads to an increased diameter of renal tubules and to renal cysts.

Mutations in the PKD2 gene cause autosomal-dominant polycystic kidney disease but the physiological role of polycystin-2, the protein product of PKD2, remains elusive. Polycystin-2 belongs to the transient receptor potential (TRP) family of non-selective cation channels. To test the hypothesis that altered ion channel properties of polycystin-2 compromise its putative role in a control circuit controlling lumen formation of renal tubular structures, we generated a mouse model in which we exchanged the pore loop of polycystin-2 with that of the closely related cation channel polycystin-2L1 (encoded by PKD2L1), thereby creating the protein polycystin-2poreL1. Functional characterization of this mutant channel in Xenopus laevis oocytes demonstrated that its electrophysiological properties differed from those of polycystin-2 and instead resembled the properties of polycystin-2L1, in particular regarding its permeability for Ca2+ ions. Homology modeling of the ion translocation pathway of polycystin-2poreL1 argues for a wider pore in polycystin-2poreL1 than in polycystin-2. In Pkd2poreL1 knock-in mice in which the endogenous polycystin-2 protein was replaced by polycystin-2poreL1 the diameter of collecting ducts was increased and collecting duct cysts developed in a strain-dependent fashion.

Cancer-associated cells release citrate to support tumour metastatic progression.

Citrate is important for lipid synthesis and epigenetic regulation in addition to ATP production. We have previously reported that cancer cells import extracellular citrate via the pmCiC transporter to support their metabolism. Here, we show for the first time that citrate is supplied to cancer by cancer-associated stroma (CAS) and also that citrate synthesis and release is one of the latter's major metabolic tasks. Citrate release from CAS is controlled by cancer cells through cross-cellular communication. The availability of citrate from CAS regulated the cytokine profile, metabolism and features of cellular invasion. Moreover, citrate released by CAS is involved in inducing cancer progression especially enhancing invasiveness and organ colonisation. In line with the in vitro observations, we show that depriving cancer cells of citrate using gluconate, a specific inhibitor of pmCiC, significantly reduced the growth and metastatic spread of human pancreatic cancer cells in vivo and muted stromal activation and angiogenesis. We conclude that citrate is supplied to tumour cells by CAS and citrate uptake plays a significant role in cancer metastatic progression.

L amino acid transporter structure and molecular bases for the asymmetry of substrate interaction.

L-amino acid transporters (LATs) play key roles in human physiology and are implicated in several human pathologies. LATs are asymmetric amino acid exchangers where the low apparent affinity cytoplasmic side controls the exchange of substrates with high apparent affinity on the extracellular side. Here, we report the crystal structures of an LAT, the bacterial alanine-serine-cysteine exchanger (BasC), in a non-occluded inward-facing conformation in both apo and substrate-bound states. We crystallized BasC in complex with a nanobody, which blocks the transporter from the intracellular side, thus unveiling the sidedness of the substrate interaction of BasC. Two conserved residues in human LATs, Tyr 236 and Lys 154, are located in equivalent positions to the Na1 and Na2 sites of sodium-dependent APC superfamily transporters. Functional studies and molecular dynamics (MD) calculations reveal that these residues are key for the asymmetric substrate interaction of BasC and in the homologous human transporter Asc-1.

Arginine-rich cell-penetrating peptides induce membrane multilamellarity and subsequently enter via formation of a fusion pore.

Arginine-rich cell-penetrating peptides do not enter cells by directly passing through a lipid membrane; they instead passively enter vesicles and live cells by inducing membrane multilamellarity and fusion. The molecular picture of this penetration mode, which differs qualitatively from the previously proposed direct mechanism, is provided by molecular dynamics simulations. The kinetics of vesicle agglomeration and fusion by an iconic cell-penetrating peptide-nonaarginine-are documented via real-time fluorescence techniques, while the induction of multilamellar phases in vesicles and live cells is demonstrated by a combination of electron and fluorescence microscopies. This concert of experiments and simulations reveals that the identified passive cell penetration mechanism bears analogy to vesicle fusion induced by calcium ions, indicating that the two processes may share a common mechanistic origin.

Extracellular Citrate Affects Critical Elements of Cancer Cell Metabolism and Supports Cancer Development In Vivo.

Glycolysis and fatty acid synthesis are highly active in cancer cells through cytosolic citrate metabolism, with intracellular citrate primarily derived from either glucose or glutamine via the tricarboxylic acid cycle. We show here that extracellular citrate is supplied to cancer cells through a plasma membrane-specific variant of the mitochondrial citrate transporter (pmCiC). Metabolomic analysis revealed that citrate uptake broadly affected cancer cell metabolism through citrate-dependent metabolic pathways. Treatment with gluconate specifically blocked pmCiC and decreased tumor growth in murine xenografts of human pancreatic cancer. This treatment altered metabolism within tumors, including fatty acid metabolism. High expression of pmCiC was associated with invasion and advanced tumor stage across many human cancers. These findings support the exploration of extracellular citrate transport as a novel potential target for cancer therapy.Significance: Uptake of extracellular citrate through pmCiC can be blocked with gluconate to reduce tumor growth and to alter metabolic characteristics of tumor tissue. Cancer Res; 78(10); 2513-23. ©2018 AACR.

Beyond Amyloid - Widening the View on Alzheimer's Disease.

For 25 years, the amyloid cascade hypothesis, based on the finding that mutations in the amyloid precursor protein are closely linked to familial forms of Alzheimer's disease (AD), dominated the research on this disease. Recent failures of clinical anti-amyloidogenic trials, however, substantially support the reasoning (i) that the pathomechanisms that trigger familial AD, namely the generation, aggregation, and deposition of amyloid beta, cannot necessarily be extrapolated to sporadic cases and (ii) that amyloid beta represents a prominent histopathological feature in AD but not its exclusive causative factor. In autumn 2016, the Volkswagen Foundation hosted the Herrenhausen Symposium 'Beyond Amyloid - Widening the View on Alzheimer's Disease' in Hannover, Germany, to bring together current knowledge on cellular and molecular processes that contribute to AD pathogenesis independent of or alongside with the amyloid biochemistry. The following mini review series was authored by key speakers at the meeting, and highlights some of the mechanisms potentially involved in AD etiology that provide alternative viewpoints and mechanisms beyond the amyloid cascade hypothesis. This article is part of the series "Beyond Amyloid".

Role of N-glycosylation in renal betaine transport.

The osmolyte and folding chaperone betaine is transported by the renal Na(+)-coupled GABA (γ-aminobutyric acid) symporter BGT-1 (betaine/GABA transporter 1), a member of the SLC6 (solute carrier 6) family. Under hypertonic conditions, the transcription, translation and plasma membrane (PM) insertion of BGT-1 in kidney cells are significantly increased, resulting in elevated betaine and GABA transport. Re-establishing isotonicity involves PM depletion of BGT-1. The molecular mechanism of the regulated PM insertion of BGT-1 during changes in osmotic stress is unknown. In the present study, we reveal a link between regulated PM insertion and N-glycosylation. Based on homology modelling, we identified two sites (Asn(171) and Asn(183)) in the extracellular loop 2 (EL2) of BGT-1, which were investigated with respect to trafficking, insertion and transport by immunogold-labelling, electron microscopy (EM), mutagenesis and two-electrode voltage clamp measurements in Xenopus laevis oocytes and uptake of radiolabelled substrate into MDCK (Madin-Darby canine kidney) and HEK293 (human embryonic kidney) cells. Trafficking and PM insertion of BGT-1 was clearly promoted by N-glycosylation in both oocytes and MDCK cells. Moreover, association with N-glycans at Asn(171) and Asn(183) contributed equally to protein activity and substrate affinity. Substitution of Asn(171) and Asn(183) by aspartate individually caused no loss of BGT-1 activity, whereas the double mutant was inactive, suggesting that N-glycosylation of at least one of the sites is required for function. Substitution by alanine or valine at either site caused a dramatic loss in transport activity. Furthermore, in MDCK cells PM insertion of N183D was no longer regulated by osmotic stress, highlighting the impact of N-glycosylation in regulation of this SLC6 transporter.

Mutation of a single threonine in the cytoplasmic NH2 terminus disrupts trafficking of renal betaine-GABA transporter 1 during hypertonic stress.

Betaine is an important osmolyte and is, compared with other organs, much more abundant in the kidneys, where it enters cells in the medulla by betaine-GABA transporter 1 (BGT1) to balance osmoregulation in the countercurrent system. In wild-type (wt-)BGT1-expressing oocytes, GABA-mediated currents were diminished by preincubation of oocytes with 100 nM PMA or 5 µM dioctanoyl-sn-glycerol, activators of PKC, whereas the application of staurosporine before the application of dioctanoyl-sn-glycerol restored the response to GABA. Four potential phosphorylation sites on BGT1 were mutated to alanine by site-directed mutagenesis. Three mutants (T235A, S428A, and S564A) evoked GABA currents comparable in magnitude to currents observed in wt-BGT1-expressing oocytes, whereas GABA currents in T40A were barely detectable. Uptake of [(3)H]GABA was also determined in human embryonic kidney-293 cells expressing enhanced green fluorescent protein (EGFP)-tagged BGT1 with the same mutations. T235A, S428A, and S564A showed upregulation of GABA uptake after hypertonic stress and downregulation by PMA similar to EGFP-wt-BGT1. In contrast, T40A did not respond to either hypertonicity or PMA. Confocal microscopy of the EGFP-BGT1 mutants expressed in Madin-Darby canine kidney cells revealed that T40A was present in the cytoplasm after 24 h of hypertonic stress. whereas the other mutants and EGFP-wt-BGT1 were in the plasma membrane. All mutants, including T40A, comigrated with wt-BGT1 on Western blots, suggesting that they are full-length proteins. T40A, however, cannot be phosphorylated, as revealed using a specific anti-phosphoantibody, and, therefore, T40 may be important for the trafficking and insertion of BGT1 in the plasma membrane.

Identification of an osmo-dependent and an osmo-independent choline transporter in Acinetobacter baylyi: implications in osmostress protection and metabolic adaptation.

Members of the genus Acinetobacter are well known for their metabolic versatility that allows them to adapt to different ecological niches. In previous studies, we have demonstrated that Acinetobacter baylyi ADP1 can cope with high salinities by uptake and accumulation of the well-known compatible solute glycine betaine. Here, we demonstrate that addition of choline restores growth at high salinities. We further show that choline was actively taken up by the cells and converted to glycine betaine. Uptake of choline was induced by high salinity and the presence of choline in the growth medium. At high salinities, glycine betaine was accumulated in the cells whereas in the absence of osmotic stress it was exported. Inspection of the genome sequence followed by mutant studies led to the identification of two genes encoding secondary transporters (BetT1 and BetT2) of the betaine-choline-carnitine transporter (BCCT) family. The BetT1 transporter lacks an extended C-terminal domain usually found in osmoregulated choline BCCTs. BetT1 was found to facilitate osmolarity-independent choline transport most likely by a uniport mechanism. We propose that BetT1 does not primarily function in osmoadaptation but might play a role in metabolic adaptation to choline-rich environments.

Structural evidence for functional lipid interactions in the betaine transporter BetP.

Bilayer lipids contribute to the stability of membrane transporters and are crucially involved in their proper functioning. However, the molecular knowledge of how surrounding lipids affect membrane transport is surprisingly limited and despite its general importance is rarely considered in the molecular description of a transport mechanism. One reason is that only few atomic resolution structures of channels or transporters reveal a functional interaction with lipids, which are difficult to detect in X-ray structures per se. Overcoming these difficulties, we report here on a new structure of the osmotic stress-regulated betaine transporter BetP in complex with anionic lipids. This lipid-associated BetP structure is important in the molecular understanding of osmoregulation due to the strong dependence of activity regulation in BetP on the presence of negatively charged lipids. We detected eight resolved palmitoyl-oleoyl phosphatidyl glycerol (PG) lipids mimicking parts of the membrane leaflets and interacting with key residues in transport and regulation. The lipid-protein interactions observed here in structural detail in BetP provide molecular insights into the role of lipids in osmoregulated secondary transport.

Results of dynamic pedobarography following surgically treated intra-articular calcaneal fractures.

OBJECTIVE: To investigate the clinical outcome and gait analysis findings by dynamic pedobarography in patients following surgically treated single, closed, dislocated intra-articular calcaneal fractures. DESIGN: Retrospective single-center study with 26 patients. The average follow-up period was 34 months (range 18-61 months). We used the Zwipp score and a score based on a visual analog scale (VAS) to assess the subjective and objective clinical outcome. Dynamic pedobarography (EMED-M, 38 × 42 cm, four sensors per square centimeter, 50 Hz; Novel GmbH., Munich, Germany) was performed to retrieve gait patterns. Analysis was performed using the Emed-Software (Novel GmbH., Munich, Germany). RESULTS: For the Zwipp score (±200 points), the average was +54.4 points (±48.2); for the VAS score (0-100 points), the average was 58.3 points (±24.3). There was limited mobility in the upper and lower ankle joint. Pedobarography showed a clearly disturbed gait with increased pressure for the fractured side (157 vs. 119 kPa) in the midfoot region (71.8 vs. 68 kPa) and under fifth metatarsal bone (234 vs. 160 kPa). The gait line was lateralized. The force-time-integral (fractured vs. healthy side) showed significant differences for the medial (18 vs. 7 N s) and lateral (61 vs. 36 N s) midfoot region. CONCLUSIONS: We found only an average clinical outcome and clear pathological gait patterns in our cohort with lateralization of the gait line.

Amino acid secondary transporters: toward a common transport mechanism.

Solute carriers (SLC) that transport amino acids are key players in health and diseases in humans. Their prokaryotic relatives are often involved in essential physiological processes in microorganisms, e.g. in homeostasis and acidic/osmotic stress response. High-resolution X-ray structures of the sequence-unrelated amino acid transporters unraveled a striking structural similarity between carriers, which were formerly assigned to different families. The highly conserved fold is characterized by two inverted structural repeats of five transmembrane helices each and indicates common mechanistic transport concepts if not an evolutionary link among a large number of amino acid transporters. Therefore, these transporters are classified now into the structural amino acid-polyamine-organocation superfamily (APCS). The APCS includes among others the mammalian SLC6 transporters and the heterodimeric SLC7/SLC3 transporters. However, it has to be noted that the APCS is not limited entirely to amino acid transporters but contains also transporters for, e.g. amino acid derivatives and sugars. For instance, the betaine-choline-carnitine transporter family of bacterial activity-regulated Na(+)- and H(+)-coupled symporters for glycine betaine and choline is also part of this second largest structural superfamily. The APCS fold provides different possibilities to transport the same amino acid. Arginine can be transported by an H(+)-coupled symport or by antiport mechanism in exchange against agmatine for example. The convergence of the mechanistic concept of transport under comparable physiological conditions allows speculating if structurally unexplored amino acid transporters, e.g. the members of the SLC36 and SLC38 family, belong to the APCS, too. In the kidney, which is an organ that depends critically on the regulated amino acid transport, these different SLC transporters have to work together to account for proper function. Here, we will summarize the basic concepts of Na(+)- and H(+)-coupled amino acid symport and amino acid-product antiport in the light of the respective physiological requirements.

Alternating-access mechanism in conformationally asymmetric trimers of the betaine transporter BetP.

Betaine and Na(+) symport has been extensively studied in the osmotically regulated transporter BetP from Corynebacterium glutamicum, a member of the betaine/choline/carnitine transporter family, which shares the conserved LeuT-like fold of two inverted structural repeats. BetP adjusts its transport activity by sensing the cytoplasmic K(+) concentration as a measure for hyperosmotic stress via the osmosensing carboxy-terminal domain. BetP needs to be in a trimeric state for communication between individual protomers through several intratrimeric interaction sites. Recently, crystal structures of inward-facing BetP trimers have contributed to our understanding of activity regulation on a molecular level. Here we report new crystal structures, which reveal two conformationally asymmetric BetP trimers, capturing among them three distinct transport states. We observe a total of four new conformations at once: an outward-open apo and an outward-occluded apo state, and two closed transition states--one in complex with betaine and one substrate-free. On the basis of these new structures, we identified local and global conformational changes in BetP that underlie the molecular transport mechanism, which partially resemble structural changes observed in other sodium-coupled LeuT-like fold transporters, but show differences we attribute to the osmolytic nature of betaine, the exclusive substrate specificity and the regulatory properties of BetP.

Role of bundle helices in a regulatory crosstalk in the trimeric betaine transporter BetP.

The Na(+)-coupled betaine symporter BetP regulates transport activity in response to hyperosmotic stress only in its trimeric state, suggesting a regulatory crosstalk between individual protomers. BetP shares the overall fold of two inverted structurally related five-transmembrane (TM) helix repeats with the sequence-unrelated Na(+)-coupled symporters LeuT, vSGLT, and Mhp1, which are neither trimeric nor regulated in transport activity. Conformational changes characteristic for this transporter fold involve the two first helices of each repeat, which form a four-TM-helix bundle. Here, we identify two ionic networks in BetP located on both sides of the membrane that might be responsible for BetP's unique regulatory behavior by restricting the conformational flexibility of the four-TM-helix bundle. The cytoplasmic ionic interaction network links both first helices of each repeat in one protomer to the osmosensing C-terminal domain of the adjacent protomer. Moreover, the periplasmic ionic interaction network conformationally locks the four-TM-helix bundle between the same neighbor protomers. By a combination of site-directed mutagenesis, cross-linking, and betaine uptake measurements, we demonstrate how conformational changes in individual bundle helices are transduced to the entire bundle by specific inter-helical interactions. We suggest that one purpose of bundle networking is to assist crosstalk between protomers during transport regulation by specifically modulating the transition from outward-facing to inward-facing state.

Locating an extracellular K+-dependent interaction site that modulates betaine-binding of the Na+-coupled betaine symporter BetP.

BetP, a trimeric Na(+)-coupled betaine symporter, senses hyperosmotic stress via its cytoplasmic C-terminal domain and regulates transport activity in dependence of the cytoplasmic K(+)-concentration. This transport regulation of BetP depends on a sophisticated interaction network. Using single-molecule force spectroscopy we structurally localize and quantify these interactions changing on K(+)-dependent transport activation and substrate-binding. K(+) significantly strengthened all interactions, modulated lifetimes of functionally important structural regions, and increased the mechanical rigidity of the symporter. Substrate-binding could modulate, but not establish most of these K(+)-dependent interactions. A pronounced effect triggered by K(+) was observed at the periplasmic helical loop EH2. Tryptophan quenching experiments revealed that elevated K(+)-concentrations akin to those BetP encounters during hyperosmotic stress trigger the formation of a periplasmic second betaine-binding (S2) site, which was found to be at a similar position reported previously for the BetP homologue CaiT. In BetP, the presence of the S2 site strengthened the interaction between EH2, transmembrane α-helix 12 and the K(+)-sensing C-terminal domain resulting in a K(+)-dependent cooperative betaine-binding.