hitin is a linear polymer of β-(1,4)-linked d-glucosamine (GlcNAc) and the second most abundant n... more hitin is a linear polymer of β-(1,4)-linked d-glucosamine (GlcNAc) and the second most abundant naturally occurring polymer on Earth, after cellulose, with annual biosynthesis of more than 100,000,000,000 tons 1. Chitin is an essential constituent of fungal cell walls and the exoskeletons of crustaceans, mollusks, and insects 2. Chitin and chitosan have also been widely studied for biomedical applications such as polymeric tissue and nanocarrier engineering 3,4. Chitin is synthesized by integral membrane Chss. Because chitin and Chs are not present in vertebrates and plants, Chs has been pursued as the target for fungicides, insecticides, and acaricides. Fungal infections pose growing threats to human health and agricultural economics worldwide. In particular, fungal coinfections of patients with COVID-19 substantially increase mortality rates 5-8. Unfortunately, there are limited classes of effective antifungal drugs while drug resistance is on the rise 9,10. Development of new antifungals is challenging due to the similarities in cellular machineries between fungi and hosts. Chs is one of the few targets whose inhibition causes highly selective growth inhibition or death of fungi without harming vertebrates or plants. Chss are conserved throughout the fungal kingdom, including but not limited to yeast, molds, and all species of mushrooms and toadstools. Chs is a membrane-embedded processive glycosyltransferase 11 , and catalyzes homopolymerization of GlcNAc using UDP-GlcNAc as substrate 12 to produce chitin, an essential polysaccharide component of the fungal cell wall 13. Chs also mediates the extrusion of the polymer product across the membrane to the extracellular side. A naturally occurring Chs inhibitor, polyoxin D, has been widely used as a safe fungicide in agriculture 14. Nikkomycin Z has been clinically investigated to treat endemic fungal infectious disease (coccidioidomycosis) 15,16. However, the development of polyoxin D and nikkomycin Z beyond their current applications has not been realized because many fungal pathogens contain Chss that are less sensitive to nikkomycins and polyoxins 17 and structural and mechanistic insights into their action on Chs are lacking. Here, we report atomic-resolution structures and structure-guided functional studies of Chs2 from Candida albicans, the leading cause of invasive fungal infections in humans. The results provide the structural basis of Chs regulation and inhibition by antifungal agents nikkomycin Z and polyoxin D and offer an unexplored avenue for the development of antifungal drugs 9. Results Structure determination. There are seven different classes of Chs in fungi, of which Class 1 has been identified as the target of nikkomycins and polyoxins. CaChs2 (Class 1) was chosen as our target for structural analysis because C. albicans is one of the leading pathological fungi in humans. CaChs2 shares 70-90% sequence identity with other Class 1 fungal Chss, 50-70% sequence identity with other classes of fungal Chss, and ~35% sequence identity to insect Chss (Supplementary Fig. 1). We heterologously expressed CaChs2 in insect cells and purified it to homogeneity (Extended Data Fig. 1a). The purified recombinant CaChs2 catalyzed formation of water-insoluble chitin polymers from UDP-GlcNAc with K m = 6.0 ± 0.7 mM and k cat = 8.6 ± 2.7 min −1 (Fig. 1a, Extended Data Fig. 1b and Supplementary Table 1). We used FTIR spectroscopy to demonstrate that the produced chitin polymer adopts the α-chitin configuration (Fig. 1b), consistent with the form of chitin found in fungal cell wall. Mg 2+ is essential for CaChs2 activity (Fig. 1c). The purified CaChs2 is inhibited by nikkomycin Z and polyoxin D with K i = 1.5 ± 0.5 and 3.2 ± 1.4 μM, respectively (Fig. 1d and Supplementary Table 1). Using isothermal titration calorimetry (ITC), we measured the K d of nikkomycin Z for CaChs2 to be ~190 nM (Extended Data Fig. 1c). These observations are consistent with the previous characterization of the crude preparation of CaChs2 18 and its homolog in Saccharomyces cerevisiae (ScChs1) 19 , suggesting successful preparation of physiologically relevant CaChs2. We determined four single-particle cryo-EM reconstructions of CaChs2 (Extended Data Figs. 2-4 and Table 1): (1) the ligand-free state resolved to ~2.95 Å, (2) in complex with UDP-GlcNAc and Mg 2+ to ~3.02 Å, (3) in complex with nikkomycin Z to ~3.19 Å, and (4) in complex with polyoxin D to ~3.15 Å. CaChs2 forms a domain-swapped homodimer (Fig. 1e,f) with each protomer composed of an N-terminal cytoplasmic glycosyltransferase domain
Members of the nucleobase/ascorbic acid transporter (NAT) gene family are found in all kingdoms o... more 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 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 substrate-binding domain (core domain) and an interface 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 ro...
Mammalian peptide transporters, PepT1 and PepT2, mediate uptake of a wide variety of di- and tri-... more Mammalian peptide transporters, PepT1 and PepT2, mediate uptake of a wide variety of di- and tri-peptides and are essential for the absorption of dietary peptides in the digestive tract and the recovery of peptides in renal filtrate. PepT also mediates absorption of many drugs and prodrugs to enhance their bioavailability. PepT has 12 transmembrane (TM) helices that fold into two domains, the N-terminal domain (NTD, TM1-6) and C-terminal domain (CTD, TM7-12), and a large extracellular domain (ECD) of ∼200 amino acids between TM9 and TM10. It is known that peptide transport involves large motions of the N- and C-domains, but the role of ECD remains unclear. Here we report the structure of PepT1 from Equus caballus (horse) determined by cryo-electron microscopy. The structure shows that ECD interacts with TM1 and bridges the N- and C-domains. Deletion of the ECD or mutations to the TM1-ECD interface both impair the transport activity. These results demonstrate a role of ECD in structu...
Cyclic nucleotide-gated (CNG) channels convert cyclic nucleotide binding and unbinding into elect... more Cyclic nucleotide-gated (CNG) channels convert cyclic nucleotide 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 C. elegans cGMP-activated channel TAX-4 reconstituted in Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
Ferroportin is an iron exporter essential for releasing cellular iron into circulation. Ferroport... more 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 trans...
SummaryHuman diacylglycerol O-acyltransferase-1 (hDGAT1) synthesizes triacylglycerides and is req... more SummaryHuman diacylglycerol O-acyltransferase-1 (hDGAT1) synthesizes triacylglycerides and is required for dietary fat absorption and fat storage. The lack of 3-dimensional structure has limited our understanding of substrate recognition and mechanism of catalysis, and hampers rational targeting of hDGAT1 for therapeutic purposes. Here we present the structure of hDGAT1 in complex with a substrate oleoyl Coenzyme A at 3.1 Å resolution. hDGAT1 forms a homodimer and each protomer has nine transmembrane helices that carve out a hollow chamber in the lipid bilayer. The chamber encloses highly conserved catalytic residues and has separate entrances for the two substrates fatty acyl Coenzyme A and diacylglycerol. The N-terminus of hDGAT1 makes extensive interactions with the neighboring protomer, and is required for enzymatic activity.
Proceedings of the National Academy of Sciences of the United States of America, Jun 5, 2018
The phosphoenolpyruvate-dependent phosphotransferase system (PTS) transports sugar into bacteria ... more 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...
Enzyme IIC (EIIC) is a membrane-embedded sugar transport protein that is part of the phosphoenolp... more 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 ...
The phosphoenolpyruvate:carbohydrate phosphotransferase systems (PTS) are found in bacteria, wher... more The phosphoenolpyruvate:carbohydrate phosphotransferase systems (PTS) 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 inwardfacing 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. Additionally, 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.
regarding how the energy is converted into useful work. To gain insight into the ion-coupled tran... more regarding how the energy is converted into useful work. To gain insight into the ion-coupled transport process, we applied a plethora of biophysical techniques including solid-state and solution NMR spectroscopy in conjunction with resistance assays to the drug transporter EmrE from E. coli. EmrE is an anti-parallel homodimer from the small multidrug resistance (SMR) family that effluxes a wide variety of quaternary cationic compounds and has served as a model for ion-coupled transport. We carried out pH titrations using NMR spectroscopy to reveal an elevated acid dissociation constant for a conserved and membrane embedded glutamate residue that is responsible for conferring drug resistance in E. coli. Furthermore, we found that the acid/base chemistry at this anionic residue was responsible for regulating the global dynamics of the transporter based on the protonation state. The implication of this result is that an open conformation of EmrE in the presence of a pH gradient would be oriented toward the higher pH side of the membrane. Indeed, tryptophan fluorescence experiments acquired in lipid bilayers strongly validate this interpretation. Finally, protonation at E14 showed long-range chemical shift perturbations far removed from the drug-binding pocket, which is evidence of an intricate allosteric network involving both concerted conformational changes to the transmembrane helices and loops that are involved in regulating the inward-open to outward-open structural switch.
HBV capsid assembly can inhibit the virus growth. The HBV capsid proteins assemble via tetramer i... more HBV capsid assembly can inhibit the virus growth. The HBV capsid proteins assemble via tetramer intermediates, with two dimer subunits and a pocket between them. Several compounds can bind to this pocket and alter orientation of the two subunits, which in turn inhibits virus growth by either accelerating the capsid assembly, or causing the proteins to assemble into non-capsid linear polymers. However, a greater therapeutic potential could be achieved by rationally designing such compounds. Here, we propose HBV capsid inhibitors from combined molecular dynamics and docking methodologies. We targeted the limiting step in the capsid formation of HBV, which is formation of a hexamer from a tetramer. Microsecondlong tetramer simulations showed slow motions of the two dimer subunits and large fluctuations in the size of the pocket between them. Based on principal component analysis of the tetramer motion, we selected a structure that was unfavorable for trimer formation and had a larger pocket volume compared to the assembled structure. In order to find potential inhibitors we docked over 100,000 compounds to the pocket in the selected structure. The top candidate compounds were selected based on docking score and surface area. The drug-protein complexes were further evaluated with molecular dynamics simulations.
GB3 and Ubiquitin, when bound to AuNPs. We find no significant changes in slow HDX rates (5-300 m... more GB3 and Ubiquitin, when bound to AuNPs. We find no significant changes in slow HDX rates (5-300 min), suggesting that AuNP-induced structural changes are small for these two proteins. Together, these results support a model where most of a protein's native contacts are preserved upon adsorption, although larger changes may occur over long timescales.
hitin is a linear polymer of β-(1,4)-linked d-glucosamine (GlcNAc) and the second most abundant n... more hitin is a linear polymer of β-(1,4)-linked d-glucosamine (GlcNAc) and the second most abundant naturally occurring polymer on Earth, after cellulose, with annual biosynthesis of more than 100,000,000,000 tons 1. Chitin is an essential constituent of fungal cell walls and the exoskeletons of crustaceans, mollusks, and insects 2. Chitin and chitosan have also been widely studied for biomedical applications such as polymeric tissue and nanocarrier engineering 3,4. Chitin is synthesized by integral membrane Chss. Because chitin and Chs are not present in vertebrates and plants, Chs has been pursued as the target for fungicides, insecticides, and acaricides. Fungal infections pose growing threats to human health and agricultural economics worldwide. In particular, fungal coinfections of patients with COVID-19 substantially increase mortality rates 5-8. Unfortunately, there are limited classes of effective antifungal drugs while drug resistance is on the rise 9,10. Development of new antifungals is challenging due to the similarities in cellular machineries between fungi and hosts. Chs is one of the few targets whose inhibition causes highly selective growth inhibition or death of fungi without harming vertebrates or plants. Chss are conserved throughout the fungal kingdom, including but not limited to yeast, molds, and all species of mushrooms and toadstools. Chs is a membrane-embedded processive glycosyltransferase 11 , and catalyzes homopolymerization of GlcNAc using UDP-GlcNAc as substrate 12 to produce chitin, an essential polysaccharide component of the fungal cell wall 13. Chs also mediates the extrusion of the polymer product across the membrane to the extracellular side. A naturally occurring Chs inhibitor, polyoxin D, has been widely used as a safe fungicide in agriculture 14. Nikkomycin Z has been clinically investigated to treat endemic fungal infectious disease (coccidioidomycosis) 15,16. However, the development of polyoxin D and nikkomycin Z beyond their current applications has not been realized because many fungal pathogens contain Chss that are less sensitive to nikkomycins and polyoxins 17 and structural and mechanistic insights into their action on Chs are lacking. Here, we report atomic-resolution structures and structure-guided functional studies of Chs2 from Candida albicans, the leading cause of invasive fungal infections in humans. The results provide the structural basis of Chs regulation and inhibition by antifungal agents nikkomycin Z and polyoxin D and offer an unexplored avenue for the development of antifungal drugs 9. Results Structure determination. There are seven different classes of Chs in fungi, of which Class 1 has been identified as the target of nikkomycins and polyoxins. CaChs2 (Class 1) was chosen as our target for structural analysis because C. albicans is one of the leading pathological fungi in humans. CaChs2 shares 70-90% sequence identity with other Class 1 fungal Chss, 50-70% sequence identity with other classes of fungal Chss, and ~35% sequence identity to insect Chss (Supplementary Fig. 1). We heterologously expressed CaChs2 in insect cells and purified it to homogeneity (Extended Data Fig. 1a). The purified recombinant CaChs2 catalyzed formation of water-insoluble chitin polymers from UDP-GlcNAc with K m = 6.0 ± 0.7 mM and k cat = 8.6 ± 2.7 min −1 (Fig. 1a, Extended Data Fig. 1b and Supplementary Table 1). We used FTIR spectroscopy to demonstrate that the produced chitin polymer adopts the α-chitin configuration (Fig. 1b), consistent with the form of chitin found in fungal cell wall. Mg 2+ is essential for CaChs2 activity (Fig. 1c). The purified CaChs2 is inhibited by nikkomycin Z and polyoxin D with K i = 1.5 ± 0.5 and 3.2 ± 1.4 μM, respectively (Fig. 1d and Supplementary Table 1). Using isothermal titration calorimetry (ITC), we measured the K d of nikkomycin Z for CaChs2 to be ~190 nM (Extended Data Fig. 1c). These observations are consistent with the previous characterization of the crude preparation of CaChs2 18 and its homolog in Saccharomyces cerevisiae (ScChs1) 19 , suggesting successful preparation of physiologically relevant CaChs2. We determined four single-particle cryo-EM reconstructions of CaChs2 (Extended Data Figs. 2-4 and Table 1): (1) the ligand-free state resolved to ~2.95 Å, (2) in complex with UDP-GlcNAc and Mg 2+ to ~3.02 Å, (3) in complex with nikkomycin Z to ~3.19 Å, and (4) in complex with polyoxin D to ~3.15 Å. CaChs2 forms a domain-swapped homodimer (Fig. 1e,f) with each protomer composed of an N-terminal cytoplasmic glycosyltransferase domain
Members of the nucleobase/ascorbic acid transporter (NAT) gene family are found in all kingdoms o... more 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 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 substrate-binding domain (core domain) and an interface 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 ro...
Mammalian peptide transporters, PepT1 and PepT2, mediate uptake of a wide variety of di- and tri-... more Mammalian peptide transporters, PepT1 and PepT2, mediate uptake of a wide variety of di- and tri-peptides and are essential for the absorption of dietary peptides in the digestive tract and the recovery of peptides in renal filtrate. PepT also mediates absorption of many drugs and prodrugs to enhance their bioavailability. PepT has 12 transmembrane (TM) helices that fold into two domains, the N-terminal domain (NTD, TM1-6) and C-terminal domain (CTD, TM7-12), and a large extracellular domain (ECD) of ∼200 amino acids between TM9 and TM10. It is known that peptide transport involves large motions of the N- and C-domains, but the role of ECD remains unclear. Here we report the structure of PepT1 from Equus caballus (horse) determined by cryo-electron microscopy. The structure shows that ECD interacts with TM1 and bridges the N- and C-domains. Deletion of the ECD or mutations to the TM1-ECD interface both impair the transport activity. These results demonstrate a role of ECD in structu...
Cyclic nucleotide-gated (CNG) channels convert cyclic nucleotide binding and unbinding into elect... more Cyclic nucleotide-gated (CNG) channels convert cyclic nucleotide 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 C. elegans cGMP-activated channel TAX-4 reconstituted in Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
Ferroportin is an iron exporter essential for releasing cellular iron into circulation. Ferroport... more 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 trans...
SummaryHuman diacylglycerol O-acyltransferase-1 (hDGAT1) synthesizes triacylglycerides and is req... more SummaryHuman diacylglycerol O-acyltransferase-1 (hDGAT1) synthesizes triacylglycerides and is required for dietary fat absorption and fat storage. The lack of 3-dimensional structure has limited our understanding of substrate recognition and mechanism of catalysis, and hampers rational targeting of hDGAT1 for therapeutic purposes. Here we present the structure of hDGAT1 in complex with a substrate oleoyl Coenzyme A at 3.1 Å resolution. hDGAT1 forms a homodimer and each protomer has nine transmembrane helices that carve out a hollow chamber in the lipid bilayer. The chamber encloses highly conserved catalytic residues and has separate entrances for the two substrates fatty acyl Coenzyme A and diacylglycerol. The N-terminus of hDGAT1 makes extensive interactions with the neighboring protomer, and is required for enzymatic activity.
Proceedings of the National Academy of Sciences of the United States of America, Jun 5, 2018
The phosphoenolpyruvate-dependent phosphotransferase system (PTS) transports sugar into bacteria ... more 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...
Enzyme IIC (EIIC) is a membrane-embedded sugar transport protein that is part of the phosphoenolp... more 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 ...
The phosphoenolpyruvate:carbohydrate phosphotransferase systems (PTS) are found in bacteria, wher... more The phosphoenolpyruvate:carbohydrate phosphotransferase systems (PTS) 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 inwardfacing 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. Additionally, 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.
regarding how the energy is converted into useful work. To gain insight into the ion-coupled tran... more regarding how the energy is converted into useful work. To gain insight into the ion-coupled transport process, we applied a plethora of biophysical techniques including solid-state and solution NMR spectroscopy in conjunction with resistance assays to the drug transporter EmrE from E. coli. EmrE is an anti-parallel homodimer from the small multidrug resistance (SMR) family that effluxes a wide variety of quaternary cationic compounds and has served as a model for ion-coupled transport. We carried out pH titrations using NMR spectroscopy to reveal an elevated acid dissociation constant for a conserved and membrane embedded glutamate residue that is responsible for conferring drug resistance in E. coli. Furthermore, we found that the acid/base chemistry at this anionic residue was responsible for regulating the global dynamics of the transporter based on the protonation state. The implication of this result is that an open conformation of EmrE in the presence of a pH gradient would be oriented toward the higher pH side of the membrane. Indeed, tryptophan fluorescence experiments acquired in lipid bilayers strongly validate this interpretation. Finally, protonation at E14 showed long-range chemical shift perturbations far removed from the drug-binding pocket, which is evidence of an intricate allosteric network involving both concerted conformational changes to the transmembrane helices and loops that are involved in regulating the inward-open to outward-open structural switch.
HBV capsid assembly can inhibit the virus growth. The HBV capsid proteins assemble via tetramer i... more HBV capsid assembly can inhibit the virus growth. The HBV capsid proteins assemble via tetramer intermediates, with two dimer subunits and a pocket between them. Several compounds can bind to this pocket and alter orientation of the two subunits, which in turn inhibits virus growth by either accelerating the capsid assembly, or causing the proteins to assemble into non-capsid linear polymers. However, a greater therapeutic potential could be achieved by rationally designing such compounds. Here, we propose HBV capsid inhibitors from combined molecular dynamics and docking methodologies. We targeted the limiting step in the capsid formation of HBV, which is formation of a hexamer from a tetramer. Microsecondlong tetramer simulations showed slow motions of the two dimer subunits and large fluctuations in the size of the pocket between them. Based on principal component analysis of the tetramer motion, we selected a structure that was unfavorable for trimer formation and had a larger pocket volume compared to the assembled structure. In order to find potential inhibitors we docked over 100,000 compounds to the pocket in the selected structure. The top candidate compounds were selected based on docking score and surface area. The drug-protein complexes were further evaluated with molecular dynamics simulations.
GB3 and Ubiquitin, when bound to AuNPs. We find no significant changes in slow HDX rates (5-300 m... more GB3 and Ubiquitin, when bound to AuNPs. We find no significant changes in slow HDX rates (5-300 min), suggesting that AuNP-induced structural changes are small for these two proteins. Together, these results support a model where most of a protein's native contacts are preserved upon adsorption, although larger changes may occur over long timescales.
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