Insight into the mechanism of H+-coupled nucleobase transport.

Members of the nucleobase/ascorbic acid transporter (NAT) gene family are found in all kingdoms of life. In mammals, the concentrative uptake of ascorbic acid (vitamin C) by members of the NAT family is driven by the Na+ gradient, while the uptake of nucleobases in bacteria is powered by the H+ gradient. Here, we report the structure and function of PurTCp, a NAT family member from Colwellia psychrerythraea. The structure of PurTCp was determined to 2.80 Å resolution by X-ray crystallography. PurTCp forms a homodimer, and each protomer has 14 transmembrane segments folded into a transport domain (core domain) and a scaffold domain (gate domain). A purine base is present in the structure and defines the location of the substrate binding site. Functional studies reveal that PurTCp transports purines but not pyrimidines and that purine binding and transport is dependent on the pH. Mutation of a conserved aspartate residue close to the substrate binding site reveals the critical role of this residue in H+-dependent transport of purines. Comparison of the PurTCp structure with transporters of the same structural fold suggests that rigid-body motions of the substrate-binding domain are central for substrate translocation across the membrane.

Structural basis for inhibition and regulation of a chitin synthase from Candida albicans.

Chitin is an essential component of the fungal cell wall. Chitin synthases (Chss) catalyze chitin formation and translocation across the membrane and are targets of antifungal agents, including nikkomycin Z and polyoxin D. Lack of structural insights into the action of these inhibitors on Chs has hampered their further development to the clinic. We present the cryo-EM structures of Chs2 from Candida albicans (CaChs2) in the apo, substrate-bound, nikkomycin Z-bound, and polyoxin D-bound states. CaChs2 adopts a unique domain-swapped dimer configuration where a conserved motif in the domain-swapped region controls enzyme activity. CaChs2 has a dual regulation mechanism where the chitin translocation tunnel is closed by the extracellular gate and plugged by a lipid molecule in the apo state to prevent non-specific leak. Analyses of substrate and inhibitor binding provide insights into the chemical logic of Chs inhibition, which can guide Chs-targeted antifungal development.

Extracellular domain of PepT1 interacts with TM1 to facilitate substrate transport.

Mammalian peptide transporters, PepT1 and PepT2, mediate uptake of small peptides and are essential for their absorption. PepT also mediates absorption of many drugs and prodrugs to enhance their bioavailability. PepT has twelve transmembrane (TM) helices that fold into an N-terminal domain (NTD, TM1-6) and a C-terminal domain (CTD, TM7-12) and has a large extracellular domain (ECD) between TM9-10. It is well recognized that peptide transport requires movements of the NTD and CTD, but the role of the ECD in PepT1 remains unclear. Here we report the structure of horse PepT1 encircled in lipid nanodiscs and captured in the inward-open apo conformation. The structure shows that the ECD bridges the NTD and CTD by interacting with TM1. Deletion of ECD or mutations to the ECD-TM1 interface impairs the transport activity. These results demonstrate an important role of ECD in PepT1 and enhance our understanding of the transport mechanism in PepT1.

Structural basis of ion transport and inhibition in ferroportin.

Ferroportin is an iron exporter essential for releasing cellular iron into circulation. Ferroportin is inhibited by a peptide hormone, hepcidin. In humans, mutations in ferroportin lead to ferroportin diseases that are often associated with accumulation of iron in macrophages and symptoms of iron deficiency anemia. Here we present the structures of the ferroportin from the primate Philippine tarsier (TsFpn) in the presence and absence of hepcidin solved by cryo-electron microscopy. TsFpn is composed of two domains resembling a clamshell and the structure defines two metal ion binding sites, one in each domain. Both structures are in an outward-facing conformation, and hepcidin binds between the two domains and reaches one of the ion binding sites. Functional studies show that TsFpn is an electroneutral H+/Fe2+ antiporter so that transport of each Fe2+ is coupled to transport of two H+ in the opposite direction. Perturbing either of the ion binding sites compromises the coupled transport of H+ and Fe2+. These results establish the structural basis of metal ion binding, transport and inhibition in ferroportin and provide a blueprint for targeting ferroportin in pharmacological intervention of ferroportin diseases.

Mechanism of ligand activation of a eukaryotic cyclic nucleotide-gated channel.

Cyclic nucleotide-gated (CNG) channels convert cyclic nucleotide (CN) binding and unbinding into electrical signals in sensory receptors and neurons. The molecular conformational changes underpinning ligand activation are largely undefined. We report both closed- and open-state atomic cryo-EM structures of a full-length Caenorhabditis elegans cyclic GMP-activated channel TAX-4, reconstituted in lipid nanodiscs. These structures, together with computational and functional analyses and a mutant channel structure, reveal a double-barrier hydrophobic gate formed by two S6 amino acids in the central cavity. cGMP binding produces global conformational changes that open the cavity gate located ~52 Å away but do not alter the structure of the selectivity filter-the commonly presumed activation gate. Our work provides mechanistic insights into the allosteric gating and regulation of CN-gated and nucleotide-modulated channels and CNG channel-related channelopathies.

Structure and mechanism of human diacylglycerol O-acyltransferase 1.

Diacylglycerol O-acyltransferase 1 (DGAT1) synthesizes triacylglycerides and is required for dietary fat absorption and fat storage in humans1. DGAT1 belongs to the membrane-bound O-acyltransferase (MBOAT) superfamily, members of which are found in all kingdoms of life and are involved in the acylation of lipids and proteins2,3. How human DGAT1 and other mammalian members of the MBOAT family recognize their substrates and catalyse their reactions is unknown. The absence of three-dimensional structures also hampers rational targeting of DGAT1 for therapeutic purposes. Here we present the cryo-electron microscopy structure of human DGAT1 in complex with an oleoyl-CoA substrate. Each DGAT1 protomer has nine transmembrane helices, eight of which form a conserved structural fold that we name the MBOAT fold. The MBOAT fold in DGAT1 forms a hollow chamber in the membrane that encloses highly conserved catalytic residues. The chamber has separate entrances for each of the two substrates, fatty acyl-CoA and diacylglycerol. DGAT1 can exist as either a homodimer or a homotetramer and the two forms have similar enzymatic activity. The N terminus of DGAT1 interacts with the neighbouring protomer and these interactions are required for enzymatic activity.

Structure of an EIIC sugar transporter trapped in an inward-facing conformation.

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) transports sugar into bacteria and phosphorylates the sugar for metabolic consumption. The PTS is important for the survival of bacteria and thus a potential target for antibiotics, but its mechanism of sugar uptake and phosphorylation remains unclear. The PTS is composed of multiple proteins, and the membrane-embedded Enzyme IIC (EIIC) component transports sugars across the membrane. Crystal structures of two members of the glucose superfamily of EIICs, bcChbC and bcMalT, were solved in the inward-facing and outward-facing conformations, and the structures suggest that sugar translocation could be achieved by movement of a structured domain that contains the sugar-binding site. However, different conformations have not been captured on the same transporter to allow precise description of the conformational changes. Here we present a crystal structure of bcMalT trapped in an inward-facing conformation by a mercury ion that bridges two strategically placed cysteine residues. The structure allows direct comparison of the outward- and inward-facing conformations and reveals a large rigid-body motion of the sugar-binding domain and other conformational changes that accompany the rigid-body motion. All-atom molecular dynamics simulations show that the inward-facing structure is stable with or without the cross-linking. The conformational changes were further validated by single-molecule Föster resonance energy transfer (smFRET). Combined, these results establish the elevator-type mechanism of transport in the glucose superfamily of EIIC transporters.

Molecular Simulation and Biochemical Studies Support an Elevator-type Transport Mechanism in EIIC.

Enzyme IIC (EIIC) is a membrane-embedded sugar transport protein that is part of the phosphoenolpyruvate-dependent phosphotransferases. Crystal structures of two members of the glucose EIIC superfamily, bcChbC in the inward-facing conformation and bcMalT in the outward-facing conformation, were previously solved. Comparing the two structures led us to the hypothesis that sugar translocation could be achieved by an elevator-type transport mechanism in which a transport domain binds to the substrate and, through rigid body motions, transports it across the membrane. To test this hypothesis and to obtain more accurate descriptions of alternate conformations of the two proteins, we first performed collective variable-based steered molecular dynamics (CVSMD) simulations starting with the two crystal structures embedded in model lipid bilayers, and steered their transport domain toward their own alternative conformation. Our simulations show that large rigid-body motions of the transport domain (55° in rotation and 8 Å in translation) lead to access of the substrate binding site to the alternate side of the membrane. H-bonding interactions between the sugar and the protein are intact, although the side chains of the binding-site residues were not restrained in the simulation. Pairs of residues in bcMalT that are far apart in the crystal structure become close to each other in the simulated model. Some of these pairs can be cross-linked by a mercury ion when mutated to cysteines, providing further support for the CVSMD-generated model. In addition, bcMalT binds to maltose with similar affinities before and after the cross-linking, suggesting that the binding site is preserved after the conformational change. In combination, these results support an elevator-type transport mechanism in EIIC.

Controllable Molecule Transport and Release by a Restorable Surface-tethered DNA nanodevice.

In this paper, we report a novel surface-tethered DNA nanodevice that may present three states and undergo conformational changes under the operation of pH. Besides, convenient regulation on the electrode surface renders the construction and operation of this DNA nanodevice restorable. To make full use of this DNA nanodevice, ferrocene (Fc) has been further employed for the fabrication of the molecular device. On one hand, the state switches of the DNA nanodevice can be characterized conveniently and reliably by the obtained electrochemical signals from Fc. On the other hand, ß-cyclodextrin-ferrocene (ß-CD-Fc) host-guest system can be introduced by Fc, which functionalizes this molecular device. Based on different electrochemical behaviors of ß-CD under different states, this DNA nanodevice can actualize directional loading, transporting and unloading of ß-CD in nanoscale. Therefore, this DNA nanodevice bares promising applications in controllable molecular transport and release, which are of great value to molecular device design.

The Structure of a Sugar Transporter of the Glucose EIIC Superfamily Provides Insight into the Elevator Mechanism of Membrane Transport.

The phosphoenolpyruvate:carbohydrate phosphotransferase systems are found in bacteria, where they play central roles in sugar uptake and regulation of cellular uptake processes. Little is known about how the membrane-embedded components (EIICs) selectively mediate the passage of carbohydrates across the membrane. Here we report the functional characterization and 2.55-Å resolution structure of a maltose transporter, bcMalT, belonging to the glucose superfamily of EIIC transporters. bcMalT crystallized in an outward-facing occluded conformation, in contrast to the structure of another glucose superfamily EIIC, bcChbC, which crystallized in an inward-facing occluded conformation. The structures differ in the position of a structurally conserved substrate-binding domain that is suggested to play a central role in sugar transport. In addition, molecular dynamics simulations suggest a potential pathway for substrate entry from the periplasm into the bcMalT substrate-binding site. These results provide a mechanistic framework for understanding substrate recognition and translocation for the glucose superfamily EIIC transporters.