Polycystin-2 (PC2), a calcium-activated cation TRP channel, is involved in diverse Ca signaling pathways. Malfunctioning Ca regulation in PC2 causes autosomal-dominant polycystic kidney disease. Here we report two cryo-EM structures of distinct channel states of full-length human PC2 in complex with lipids and cations. The structures reveal conformational differences in the selectivity filter and in the large exoplasmic domain (TOP domain), which displays differing N-glycosylation. The more open structure has one cation bound below the selectivity filter (single-ion mode, PC2), whereas multiple cations are bound along the translocation pathway in the second structure (multi-ion mode, PC2). Ca binding at the entrance of the selectivity filter suggests Ca blockage in PC2, and we observed density for the Ca-sensing C-terminal EF hand in the unblocked PC2 state. The states show altered interactions of lipids with the pore loop and TOP domain, thus reflecting the functional diversity of PC2 at different locations, owing to different membrane compositions.

Significance The crystallographic model of the Major Facilitator Superfamily (MFS) member, d -xylose permease XylE from Escherichia coli , a homologue of human d -glucose transporters, the GLUTs (SLC2), provides a structural framework for the identification and physical localization of crucial residues in transporters with medical relevance (i.e. the GLUTs). The mechanism and substrate specificity of human and prokaryotic sugar transporters are discussed by using homology modeling, molecular docking, and experimentation. Substrate-specificity determinants for XylE, GLUT1, and GLUT5 are proposed. Furthermore, concepts derived from other bacterial MFS transporters are examined for their relevance to the GLUTs by comparing conservation of critical residues. XylE mutants that mimic the characteristics of GLUT1 are tested, revealing that uniport and symport are mechanistically related.

Major facilitator superfamily (MFS) transport proteins are ubiquitous in the membranes of all living cells, and ∼25% of prokaryotic membrane transport proteins belong to this superfamily. The MFS represents the largest and most diverse group of transporters and includes members that are clinically important. A wide range of substrates is transported in many instances actively by transduction of the energy stored in an H + electrochemical gradient into a concentration gradient of substrate. MFS transporters are characterized by a deep central hydrophilic cavity surrounded by 12 mostly irregular transmembrane helices. An alternating inverted triple-helix structural symmetry within the N-and C-terminal sixhelix bundles suggests that the proteins arose by intragenic multiplication. However, despite similar features, MFS transporters share only weak sequence homology. Here, we show that rearrangement of the structural symmetry motifs in the Escherichia coli fucose permease (FucP) results in remarkable homology to lactose permease (LacY). The finding is supported by comparing the location of 34 point mutations in FucP to the location of mutants in LacY. Furthermore, in contrast to the conventional, linear sequence alignment, homologies between sugar-and H + -binding sites in the two proteins are observed. Thus, LacY and FucP likely evolved from primordial helix-triplets that formed functional transporters; however, the functional segments assembled in a different consecutive order. The idea suggests a simple, parsimonious chain of events that may have led to the enormous sequence diversity within the MFS.membrane proteins | sequence analysis

The lactose permease (LacY) of Escherichia coli, a paradigm for the major facilitator superfamily, catalyzes the coupled stoichiometric translocation of a galactopyranoside and an H + across the cytoplasmic membrane. To catalyze transport, LacY undergoes large conformational changes that allow alternating access of sugarand H + -binding sites to either side of the membrane. Despite strong evidence for an alternating access mechanism, it remains unclear how H + -and sugar-binding trigger the cascade of interactions leading to alternating conformational states. Here we used dynamic single-molecule force spectroscopy to investigate how substrate binding induces this phenomenon. Galactoside binding strongly modifies kinetic, energetic, and mechanical properties of the N-terminal 6-helix bundle of LacY, whereas the C-terminal 6-helix bundle remains largely unaffected. Within the N-terminal 6-helix bundle, the properties of helix V, which contains residues critical for sugar binding, change most radically. Particularly, secondary structures forming the N-terminal domain exhibit mechanically brittle properties in the unbound state, but highly flexible conformations in the substrate-bound state with significantly increased lifetimes and energetic stability. Thus, sugar binding tunes the properties of the N-terminal domain to initiate galactoside/H + symport. In contrast to wild-type LacY, the properties of the conformationally restricted mutant Cys154➝Gly do not change upon sugar binding. It is also observed that the single mutation of Cys154➝Gly alters intramolecular interactions so that individual transmembrane helices manifest different properties. The results support a working model of LacY in which substrate binding induces alternating conformational states and provides insight into their specific kinetic, energetic, and mechanical properties.atomic force microscopy | membrane | transport protein | membrane protein structure | membrane protein folding | membrane transport T he lactose permease of Escherichia coli (LacY) of the major facilitator superfamily (MFS) (1, 2) catalyzes the coupled stoichiometric translocation of a galactopyranoside and an H + (sugar/H + symport) (3-6). Uphill (i.e., active) symport of galactoside against a concentration gradient is achieved by transduction of free energy released from the downhill movement of H + with the electrochemical H + gradient (Δμ H + ; interior negative and/or alkaline). Conversely, because coupling between sugar and H + is obligatory, downhill galactoside transport from a high to a low sugar concentration is coupled to uphill H + transport with the generation of Δμ H +, the polarity of which depends upon the direction of the sugar concentration gradient (7-10).LacY monomers reconstituted into proteoliposomes are functional (11, 12), and X-ray crystal structures reveal 12, mostly irregular, transmembrane α-helices organized into two pseudosymmetrical 6-helix bundles surrounding a large interior hydrophilic cavity open to the cytoplasm (13-16). At the apex of the h...

Membrane protein complexes can support both the generation and utilisation of a transmembrane electrochemical proton potential ('proton-motive force'), either by transmembrane electron transfer coupled to protolytic reactions on opposite sides of the membrane or by transmembrane proton transfer. Here we provide the first evidence that both of these mechanisms are combined in the case of a specific respiratory membrane protein complex, the dihaem-containing quinol:fumarate reductase (QFR) of Wolinella succinogenes, so as to facilitate transmembrane electron transfer by transmembrane proton transfer. We also demonstrate the non-functionality of this novel transmembrane proton transfer pathway ('E-pathway') in a variant QFR where a key glutamate residue has been replaced. The 'E-pathway', discussed on the basis of the 1.78-Angstrom-resolution crystal structure of QFR, can be concluded to be essential also for the viability of pathogenic epsilon-proteobacteria such as Helicobacter pylori and is possibly relevant to proton transfer in other dihaem-containing membrane proteins, performing very different physiological functions.

Bacterial sugar symporters in the Major Facilitator Superfamily (MFS) use the H+ (and in a few cases Na+) electrochemical gradients to achieve active transport of sugar into the cell. Because a number of structures of MFS sugar symporters have been solved recently, molecular insight into the transport mechanism is possible from detailed functional analysis. We present here a comparative electrophysiological study of the lactose permease (LacY), the fucose permease (FucP) and the xylose permease (XylE), which reveals common mechanistic principles and differences. In all three symporters energetically downhill electrogenic sugar/H+ symport is observed. Comparison of the pH dependence of symport at symmetrical pH exhibits broad bell-shaped pH profiles extending over 3 to 6 pH units and a decrease at extremely alkaline pH ≥ 9.4 and at acidic to neutral pH = 4.6–7.5. The pH dependence can be described by an acidic to neutral apparent pK (pKapp) and an alkaline pKapp. Experimental evidence suggests that the alkaline pKapp is due to H+ depletion at the protonation site, while the acidic pKapp is due to inhibition of deprotonation. Since previous studies suggest that a single carboxyl group in LacY (Glu325) may be the only side chain directly involved in H+ translocation and a carboxyl side chain with similar properties has been identified in FucP (Asp46) and XylE (Asp27), the present results imply that the pK of this residue is switched during H+/sugar symport in all three symporters.

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. Uptake of extracellular citrate through pmCiC can be blocked with gluconate to reduce tumor growth and to alter metabolic characteristics of tumor tissue. .

Reconciliation of apparently contradictory experimental results obtained on the quinol:fumarate reductase, a diheme-containing respiratory membrane protein complex from Wolinella succinogenes, was previously obtained by the proposal of the so-called ''E pathway hypothesis.'' According to this hypothesis, transmembrane electron transfer via the heme groups is strictly coupled to cotransfer of protons via a transiently established pathway thought to contain the side chain of residue Glu-C180 as the most prominent component. Here we demonstrate that, after replacement of Glu-C180 with Gln or Ile by site-directed mutagenesis, the resulting mutants are unable to grow on fumarate, and the membrane-bound variant enzymes lack quinol oxidation activity. Upon solubilization, however, the purified enzymes display Ϸ1͞10 of the specific quinol oxidation activity of the wild-type enzyme and unchanged quinol Michaelis constants, K m. The refined x-ray crystal structures at 2.19 Å and 2.76 Å resolution, respectively, rule out major structural changes to account for these experimental observations. Changes in the oxidation-reduction heme midpoint potential allow the conclusion that deprotonation of Glu-C180 in the wild-type enzyme facilitates the reoxidation of the reduced high-potential heme. Comparison of solvent isotope effects indicates that a rate-limiting proton transfer step in the wild-type enzyme is lost in the Glu-C180 3 Gln variant. The results provide experimental evidence for the validity of the E pathway hypothesis and for a crucial functional role of Glu-C180.anaerobic respiration ͉ atomic model ͉ bioenergetics ͉ membrane protein ͉ succinate:quinone oxidoreductase A ccording to Peter Mitchell's chemiosmotic theory (1), the energy released upon the oxidation of electron donor substrates in both aerobic and anaerobic respiration is transiently stored in the form of an electrochemical proton potential (⌬p) across the energy-transducing membranes, which can then be used by the ATP synthase for ADP phosphorylation with inorganic phosphate. Fundamentally, there are two mechanisms by which integral membrane proteins can act as catalysts in this coupling of electron transfer reactions to the generation of a transmembrane ⌬p: the redox loop mechanism and the proton pump mechanism (2). The redox loop mechanism essentially involves transmembrane electron transfer. Reduction reactions on one side of the energytransducing membrane are associated with proton binding, whereas oxidation reactions on the opposite side of the membrane are associated with proton release. In a simple form, this mechanism is represented by the formate dehydrogenase (3) and membranebound nitrate reductase (4) enzymes of anaerobic respiration and, in a more complicated form, by the Q-cycle of the cytochrome bc 1