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The decoration of secretory glycoproteins and glycolipids with sialic acid is critical to many physiological and pathological processes. Sialyation is dependent on a continuous supply of sialic acid into Golgi organelles in the form of CMP-sialic acid. Translocation of CMP-sialic acid into Golgi is carried out by the CMP-sialic acid transporter (CST). Mutations in human CST are linked to glycosylation disorders, and CST is important for glycopathway engineering, as it is critical for sialyation efficiency of therapeutic glycoproteins. The mechanism of how CMP-sialic acid is recognized and translocated across Golgi membranes in exchange for CMP is poorly understood. Here we have determined the crystal structure of a eukaryotic CMP-sialic acid transporter in complex with CMP. We conclude that the specificity of CST for CMP-sialic acid is established by the nucleotide CMP to such an extent, they are uniquely able to work both as passive and as (secondary) active antiporters.

Elucidating the mechanism of sugar import requires a molecular understanding of how transporters couple sugar binding and gating events. Whereas mammalian glucose transporters (GLUTs) are specialists1, the hexose transporter from the malaria parasite Plasmodium falciparum PfHT12,3 has acquired the ability to transport both glucose and fructose sugars as efficiently as the dedicated glucose (GLUT3) and fructose (GLUT5) transporters. Here, to establish the molecular basis of sugar promiscuity in malaria parasites, we determined the crystal structure of PfHT1 in complex with d-glucose at a resolution of 3.6 A. We found that the sugar-binding site in PfHT1 is very similar to those of the distantly related GLUT3 and GLUT5 structures4,5. Nevertheless, engineered PfHT1 mutations made to match GLUT sugar-binding sites did not shift sugar preferences. The extracellular substrate-gating helix TM7b in PfHT1 was positioned in a fully occluded conformation, providing a unique glimpse into how sugar binding and gating are coupled. We determined that polar contacts between TM7b and TM1 (located about 15 A from d-glucose) are just as critical for transport as the residues that directly coordinate d-glucose, which demonstrates a strong allosteric coupling between sugar binding and gating. We conclude that PfHT1 has achieved substrate promiscuity not by modifying its sugar-binding site, but instead by evolving substrate-gating dynamics. Crystal structure of the Plasmodium falciparum hexose transporter PfHT1 reveals the molecular basis of its ability to transport multiple types of sugar as efficiently as the dedicated mammalian glucose and fructose transporters.

Optimising membrane protein production yields in Escherichia coli can be time- and resource-consuming. Here, we present a simple and effective Membrane protein Single shot amplification recipe: MemStar. This one-shot amplification recipe is based on the E. coli strain Lemo21(DE3), the PASM-5052 auto-induction medium and, contradictorily, an IPTG induction step. Using MemStar, production yields for most bacterial membrane proteins tested were improved to reach an average of 5mgL−1 per OD600 unit, which is significantly higher than yields obtained with other common production strategies. With MemStar, we have been able to obtain new structural information for several transporters, including the sodium/proton antiporter NapA.

Leukotriene A4 hydrolase/aminopeptidase (LTA4H) (EC 3.3.2.6) is a bifunctional zinc metalloenzyme with both an epoxide hydrolase and an aminopeptidase activity. LTA4H from the African claw toad, Xenopus laevis (xlLTA4H) has been shown to, unlike the human enzyme, convert LTA4 to two enzymatic metabolites, LTB4 and another biologically active product Δ(6)-trans-Δ(8)-cis-LTB4 (5(S),12R-dihydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid). In order to study the molecular aspect of the formation of this product we have characterized the structure and function of xlLTA4H. We solved the structure of xlLTA4H to a resolution of 2.3Å. It is a dimeric structure where each monomer has three domains with the active site in between the domains, similar as to the human structure. An important difference between the human and amphibian enzyme is the phenylalanine to tyrosine exchange at position 375. Our studies show that mutating F375 in xlLTA4H to tyrosine abolishes the formation of the LTB4 isomeric product Δ(6)-trans-Δ(8)-cis-LTB4. In an attempt to understand how one amino acid exchange leads to a new product profile as seen in the xlLTA4H, we performed a conformer analysis of the triene part of the substrate LTA4. Our results show that the Boltzmann distribution of substrate conformers correlates with the observed distribution of products. We suggest that the observed difference in product profile between the human and the xlLTA4H arises from different level of discrimination between substrate LTA4 conformers.

Radon concentrations were measured in kucha, semi-kucha and pucka houses located on the sedimentary rocks of Early Pleistocene-Pliocene Soan Formation, Pleistocene Mirpur conglomerate and recent deposits. The radon concentration was measured using CN 85 nuclear tracks etch detectors in box type dosimeters installed in the bedrooms and kitchens of the houses. It was found to vary from 14 ± 7 Bqm-3to 258 ± 28 Bqm-3with an average of 105 ± 17 Bqm-3. The average radon concentration in the kucha, semi-kucha and pucka houses was 78 ± 15 Bqm-3, 108 ± 18 Bqm-3and 108± 18 Bqm-3respectively. The increase in radon concentration from kucha to pucka houses was attributed to the type of house, differing humidity related to the Mangla Dam, temperature, bed rock geology, structures (joints, fractures and faults), aggregates and uranium-bearing sand. This study shows that the average radon level is within the accepted safe health limit (200 Bqm-3) for the inhabitants of New Mirpur Azad Kashmir, Pakistan.

Leukotriene (LT) C4 synthase (LTC4S) is an integral membrane protein that catalyzes the conjugation reaction between the fatty acid LTA4 and GSH to form the pro-inflammatory LTC4, an important mediator of asthma. Mouse models of inflammatory disorders such as asthma are key to improve our understanding of pathogenesis and potential therapeutic targets. Here, we solved the crystal structure of mouse LTC4S in complex with GSH and a product analog, S-hexyl-GSH. Furthermore, we synthesized a nM inhibitor and compared its efficiency and binding mode against the purified mouse and human isoenzymes, along with the enzymes’ steady-state kinetics. Although structural differences near the active site and along the C-terminal α-helix V suggest that the mouse and human LTC4S may function differently in vivo, our data indicate that mouse LTC4S will be a useful tool in future pharmacological research and drug development.