(2015) Crystal structure of the anion exchanger domain of human erythrocyte band 3. Science, 350 (6261). pp. 680-684. Permanent WRAP url:

The uric acid/xanthine H+ symporter, UapA, is a high-affinity purine transporter from the filamentous fungus Aspergillus nidulans. Here we present the crystal structure of a genetically stabilized version of UapA (UapA-G411VΔ1–11) in complex with xanthine. UapA is formed from two domains, a core domain and a gate domain, similar to the previously solved uracil transporter UraA, which belongs to the same family. The structure shows UapA in an inward-facing conformation with xanthine bound to residues in the core domain. Unlike UraA, which was observed to be a monomer, UapA forms a dimer in the crystals with dimer interactions formed exclusively through the gate domain. Analysis of dominant negative mutants is consistent with dimerization playing a key role in transport. We postulate that UapA uses an elevator transport mechanism likely to be shared with other structurally homologous transporters including anion exchangers and prestin.

The crystal structure of the dimeric membrane domain of human Band 3(1), the red cell chloride/bicarbonate anion exchanger 1 (AE1, SLC4A1), provides a structural context for over four decades of studies into this historic and important membrane glycoprotein. In this review, we highlight the key structural features responsible for anion binding and translocation and have integrated the following topological markers within the Band 3 structure: blood group antigens, N-glycosylation site, protease cleavage sites, inhibitor and chemical labeling sites, and the results of scanning cysteine and N-glycosylation mutagenesis. Locations of mutations linked to human disease, including those responsible for Southeast Asian ovalocytosis, hereditary stomatocytosis, hereditary spherocytosis, and distal renal tubular acidosis, provide molecular insights into their effect on Band 3 folding. Finally, molecular dynamics simulations of phosphatidylcholine self-assembled around Band 3 provide a view of this membrane protein within a lipid bilayer.

A systematic approach to the scaling and merging of data from multiple crystals in macromolecular crystallography is introduced and explained.

Antibiotic resistance is a widely spread phenomenon. One major mechanism that underlies antibiotic resistance in bacteria is the active extrusion of toxic compounds through the membrane-bound efflux pumps that are often regulated at the transcriptional level. TtgR represses the transcription of TtgABC, a key efflux pump in Pseudomonas putida, which is highly resistant to antibiotics, solvents and toxic plant secondary products. Previously we showed that TtgR is the only reported repressor that binds to different classes of natural antimicrobial compounds, which are also extruded by the efflux pump. We report here five high-resolution crystal structures of TtgR from the solvent-tolerant strain DOT-T1E, including TtgR in complex with common antibiotics and plant secondary metabolites. We provide structural basis for the unique ligand binding properties of TtgR. We identify two distinct and overlapping ligand binding sites; the first one is broader and consists of mainly hydrophobic residues, whereas the second one is deeper and contains more polar residues including Arg176, a unique residue present in the DOT-T1E strain but not in other Pseudomonas strains. Phloretin, a plant antimicrobial, can bind to both binding sites with distinct binding affinities and stoichiometries. Results on ligand binding properties of native and mutant TtgR proteins using isothermal titration calorimetry confirm the binding affinities and stoichiometries, and suggest a potential positive cooperativity between the two binding sites. The importance of Arg176 in phloretin binding was further confirmed by the reduced ability of phloretin in releasing the mutant TtgR from bound DNA compared to the native protein. The results presented here highlight the importance and versatility of regulatory systems in bacterial antibiotic resistance and open up new avenues for novel antimicrobial development.