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Structural basis for inhibition of the drug efflux pump NorA from Staphylococcus aureus

Brawley, Douglas N; Sauer, David B; Li, Jianping; Zheng, Xuhui; Koide, Akiko; Jedhe, Ganesh S; Suwatthee, Tiffany; Song, Jinmei; Liu, Zheng; Arora, Paramjit S; Koide, Shohei; Torres, Victor J; Wang, Da-Neng; Traaseth, Nathaniel J
Membrane protein efflux pumps confer antibiotic resistance by extruding structurally distinct compounds and lowering their intracellular concentration. Yet, there are no clinically approved drugs to inhibit efflux pumps, which would potentiate the efficacy of existing antibiotics rendered ineffective by drug efflux. Here we identified synthetic antigen-binding fragments (Fabs) that inhibit the quinolone transporter NorA from methicillin-resistant Staphylococcus aureus (MRSA). Structures of two NorA-Fab complexes determined using cryo-electron microscopy reveal a Fab loop deeply inserted in the substrate-binding pocket of NorA. An arginine residue on this loop interacts with two neighboring aspartate and glutamate residues essential for NorA-mediated antibiotic resistance in MRSA. Peptide mimics of the Fab loop inhibit NorA with submicromolar potency and ablate MRSA growth in combination with the antibiotic norfloxacin. These findings establish a class of peptide inhibitors that block antibiotic efflux in MRSA by targeting indispensable residues in NorA without the need for membrane permeability.
PMID: 35361990
ISSN: 1552-4469
CID: 5201392

Structural basis of ion - substrate coupling in the Na+-dependent dicarboxylate transporter VcINDY

Sauer, David B; Marden, Jennifer J; Sudar, Joseph C; Song, Jinmei; Mulligan, Christopher; Wang, Da-Neng
The Na+-dependent dicarboxylate transporter from Vibrio cholerae (VcINDY) is a prototype for the divalent anion sodium symporter (DASS) family. While the utilization of an electrochemical Na+ gradient to power substrate transport is well established for VcINDY, the structural basis of this coupling between sodium and substrate binding is not currently understood. Here, using a combination of cryo-EM structure determination, succinate binding and site-directed cysteine alkylation assays, we demonstrate that the VcINDY protein couples sodium- and substrate-binding via a previously unseen cooperative mechanism by conformational selection. In the absence of sodium, substrate binding is abolished, with the succinate binding regions exhibiting increased flexibility, including HPinb, TM10b and the substrate clamshell motifs. Upon sodium binding, these regions become structurally ordered and create a proper binding site for the substrate. Taken together, these results provide strong evidence that VcINDY's conformational selection mechanism is a result of the sodium-dependent formation of the substrate binding site.
PMID: 35551191
ISSN: 2041-1723
CID: 5214742

The ups and downs of elevator-type di-/tricarboxylate membrane transporters

Sauer, David B; Wang, Bing; Sudar, Joseph C; Song, Jinmei; Marden, Jennifer; Rice, William J; Wang, Da-Neng
The divalent anion sodium symporter (DASS) family contains both sodium-driven anion cotransporters and anion/anion exchangers. The family belongs to a broader ion transporter superfamily (ITS), which comprises 24 families of transporters, including those of AbgT antibiotic efflux transporters. The human proteins in the DASS family play major physiological roles and are drug targets. We recently determined multiple structures of the human sodium-dependent citrate transporter (NaCT) and the succinate/dicarboxylate transporter from Lactobacillus acidophilus (LaINDY). Structures of both proteins show high degrees of structural similarity to the previously determined VcINDY fold. Conservation between these DASS protein structures and those from the AbgT family indicates that the VcINDY fold represents the overall protein structure for the entire ITS. The new structures of NaCT and LaINDY are captured in the inward- or outward-facing conformations, respectively. The domain arrangements in these structures agree with a rigid body elevator-type transport mechanism for substrate translocation across the membrane. Two separate NaCT structures in complex with a substrate or an inhibitor allowed us to explain the inhibition mechanism and propose a detailed classification scheme for grouping disease-causing mutations in the human protein. Structural understanding of multiple kinetic states of DASS proteins is a first step toward the detailed characterization of their entire transport cycle.
PMID: 34403567
ISSN: 1742-4658
CID: 5066842

Structure and inhibition mechanism of the human citrate transporter NaCT

Sauer, David B; Song, Jinmei; Wang, Bing; Hilton, Jacob K; Karpowich, Nathan K; Mindell, Joseph A; Rice, William J; Wang, Da-Neng
Citrate is best known as an intermediate in the tricarboxylic acid cycle of the cell. In addition to this essential role in energy metabolism, the tricarboxylate anion also acts as both a precursor and a regulator of fatty acid synthesis1-3. Thus, the rate of fatty acid synthesis correlates directly with the cytosolic concentration of citrate4,5. Liver cells import citrate through the sodium-dependent citrate transporter NaCT (encoded by SLC13A5) and, as a consequence, this protein is a potential target for anti-obesity drugs. Here, to understand the structural basis of its inhibition mechanism, we determined cryo-electron microscopy structures of human NaCT in complexes with citrate or a small-molecule inhibitor. These structures reveal how the inhibitor-which binds to the same site as citrate-arrests the transport cycle of NaCT. The NaCT-inhibitor structure also explains why the compound selectively inhibits NaCT over two homologous human dicarboxylate transporters, and suggests ways to further improve the affinity and selectivity. Finally, the NaCT structures provide a framework for understanding how various mutations abolish the transport activity of NaCT in the brain and thereby cause epilepsy associated with mutations in SLC13A5 in newborns (which is known as SLC13A5-epilepsy)6-8.
PMID: 33597751
ISSN: 1476-4687
CID: 4788372

Structural basis for the reaction cycle of DASS dicarboxylate transporters

Sauer, David B; Trebesch, Noah; Marden, Jennifer J; Cocco, Nicolette; Song, Jinmei; Koide, Akiko; Koide, Shohei; Tajkhorshid, Emad; Wang, Da-Neng
Citrate, α-ketoglutarate and succinate are TCA cycle intermediates that also play essential roles in metabolic signaling and cellular regulation. These di- and tricarboxylates are imported into the cell by the divalent anion sodium symporter (DASS) family of plasma membrane transporters, which contains both cotransporters and exchangers. While DASS proteins transport substrates via an elevator mechanism, to date structures are only available for a single DASS cotransporter protein in a substrate-bound, inward-facing state. We report multiple cryo-EM and X-ray structures in four different states, including three hitherto unseen states, along with molecular dynamics simulations, of both a cotransporter and an exchanger. Comparison of these outward- and inward-facing structures reveal how the transport domain translates and rotates within the framework of the scaffold domain through the transport cycle. Additionally, we propose that DASS transporters ensure substrate coupling by a charge-compensation mechanism, and by structural changes upon substrate release.
PMID: 32869741
ISSN: 2050-084x
CID: 4583042

Predicting the Optimal Growth Temperatures of Prokaryotes using only Genome Derived Features

Sauer, David B; Wang, Da-Neng
Motivation/UNASSIGNED:Optimal growth temperature is a fundamental characteristic of all living organisms. Knowledge of this temperature is central to the study of a prokaryote, the thermal stability and temperature dependent activity of its genes, and the bioprospecting of its genome for thermally adapted proteins. While high throughput sequencing methods have dramatically increased the availability of genomic information, the growth temperatures of the source organisms are often unknown. This limits the study and technological application of these species and their genomes. Here, we present a novel method for the prediction of growth temperatures of prokaryotes using only genomic sequences. Results/UNASSIGNED:By applying the reverse ecology principle that an organism's genome includes identifiable adaptations to its native environment, we can predict a species' optimal growth temperature with an accuracy of 5.17 °C root-mean-square error and a coefficient of determination of 0.835. The accuracy can be further improved for specific taxonomic clades or by excluding psychrophiles. This method provides a valuable tool for the rapid calculation of organism growth temperature when only the genome sequence is known. Availability and implementation/UNASSIGNED:Source code, genomes analyzed, and features calculated are available at: Supplementary information/UNASSIGNED:Supplementary data are available at Bioinformatics online.
PMID: 30689741
ISSN: 1367-4811
CID: 3626442

Ke Hsin Kuo: A distinguished scientist and great mentor

Wang, Da-Neng; Qin, Lu-Chang
PMID: 30426341
ISSN: 1674-8018
CID: 3458622

Six-Transmembrane Epithelial Antigen of Prostate 1 (STEAP1) Has a Single b Heme and Is Capable of Reducing Metal Ion Complexes and Oxygen

Kim, Kwangsoo; Mitra, Sharmistha; Wu, Gang; Berka, Vladimir; Song, Jinmei; Yu, Ye; Poget, Sebastien; Wang, Da-Neng; Tsai, Ah-Lim; Zhou, Ming
STEAP1, six-transmembrane epithelial antigen of prostate member 1, is strongly expressed in several types of cancer cells, particularly in prostate cancer, and inhibition of its expression reduces the rate of tumor cell proliferation. However, the physiological function of STEAP1 remains unknown. Here for the first time, we purified a mammalian (rabbit) STEAP1 at a milligram level, permitting its high-quality biochemical and biophysical characterizations. We found that STEAP1 likely assembles as a homotrimer and forms a heterotrimer when co-expressed with STEAP2. Each STEAP1 protomer binds one heme prosthetic group that is mainly low-spin with a pair of histidine axial ligands, with small portions of high-spin and P450-type heme. In its ferrous state, STEAP1 is capable of reducing transition metal ion complexes of Fe3+ and Cu2+. Ferrous STEAP1 also reacts readily with O2 through an outer sphere redox mechanism. Kinetics with all three substrates are biphasic with approximately 80 and approximately 20% for the fast and slow phases, respectively, in line with its heme heterogeneity. STEAP1 retained a low level of bound FAD during purification, and the binding equilibrium constant, KD, was approximately 30 muM. These results highlight STEAP as a novel metal reductase and superoxide synthase and establish a solid basis for further research into understanding how STEAP1 activities may affect cancer progression.
PMID: 27792302
ISSN: 1520-4995
CID: 2353122

An aromatic cap seals the substrate binding site in an ECF-type S subunit for riboflavin

Karpowich, Nathan K; Song, Jinmei; Wang, Da-Neng
ECF transporters are a family of active membrane transporters for essential micronutrients, such as vitamins and trace metals. Found exclusively in archaea and bacteria, these transporters are composed of four subunits: an integral membrane substrate-binding subunit (EcfS), a transmembrane coupling subunit (EcfT), and two ATP-binding cassette ATPases (EcfA and EcfA'). We have characterized the structural basis of substrate binding by the EcfS subunit for riboflavin from T. maritima, TmRibU. TmRibU binds riboflavin with high affinity, and the protein-substrate complex is exceptionally stable in solution. The crystal structure of riboflavin-bound TmRibU reveals an electronegative binding pocket at the extracellular surface in which the substrate is completely buried. Analysis of the intermolecular contacts indicates that nearly every available substrate hydrogen bond is satisfied. A conserved aromatic residue at the extracellular end of TM5, Tyr130, caps the binding site to generate a substrate-bound occluded state, and nonconservative mutation of Tyr130 reduces the stability of this conformation. Using a novel fluorescence binding assay, we find that an aromatic residue at this position is essential for high affinity substrate binding. Comparison with other S subunit structures suggests that TM5 and Loop5-6 contain a dynamic conserved motif that plays a key role in gating substrate entry and release by S subunits of ECF transporters.
PMID: 27312125
ISSN: 1089-8638
CID: 2145272

Rapid Bioinformatic Identification of Thermostabilizing Mutations

Sauer, David B; Karpowich, Nathan K; Song, Jin Mei; Wang, Da-Neng
Ex vivo stability is a valuable protein characteristic but is laborious to improve experimentally. In addition to biopharmaceutical and industrial applications, stable protein is important for biochemical and structural studies. Taking advantage of the large number of available genomic sequences and growth temperature data, we present two bioinformatic methods to identify a limited set of amino acids or positions that likely underlie thermostability. Because these methods allow thousands of homologs to be examined in silico, they have the advantage of providing both speed and statistical power. Using these methods, we introduced, via mutation, amino acids from thermoadapted homologs into an exemplar mesophilic membrane protein, and demonstrated significantly increased thermostability while preserving protein activity.
PMID: 26445442
ISSN: 1542-0086
CID: 1793182