Structural basis for the reaction cycle of DASS dicarboxylate transporters.

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.

ATP binding drives substrate capture in an ECF transporter by a release-and-catch mechanism.

ECF transporters are a family of active transporters for vitamins. They are composed of four subunits: a membrane-embedded substrate-binding subunit (EcfS), a transmembrane coupling subunit (EcfT) and two ATP-binding-cassette ATPases (EcfA and EcfA'). We have investigated the mechanism of the ECF transporter for riboflavin from the pathogen Listeria monocytogenes, LmECF-RibU. Using structural and biochemical approaches, we found that ATP binding to the EcfAA' ATPases drives a conformational change that dissociates the S subunit from the EcfAA'T ECF module. Upon release from the ECF module, the RibU S subunit then binds the riboflavin transport substrate. We also find that S subunits for distinct substrates compete for the ATP-bound state of the ECF module. Our results explain how ECF transporters capture the transport substrate and reproduce the in vivo observations on S-subunit competition for which the family was named.

Influences on trimerization and aggregation of soluble, cleaved HIV-1 SOSIP envelope glycoprotein.

We describe methods to improve the properties of soluble, cleaved gp140 trimers of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) for use in structural studies and as immunogens. In the absence of nonionic detergents, gp140 of the KNH1144 genotype, terminating at residue 681 in gp41 (SOSIP.681), has a tendency to form higher-order complexes or aggregates, which is particularly undesirable for structure-based research. We found that this aggregation in the absence of detergent does not involve the V1, V2, or V3 variable regions of gp120. Moreover, we observed that detergent forms micelles around the membrane-proximal external region (MPER) of the SOSIP.681 gp140 trimers, whereas deletion of most of the MPER residues by terminating the gp140 at residue 664 (SOSIP.664) prevented the aggregation that otherwise occurs in SOSIP.681 in the absence of detergent. Although the MPER can contribute to trimer formation, truncation of most of it only modestly reduced trimerization and lacked global adverse effects on antigenicity. Thus, the MPER deletion minimally influenced the kinetics of the binding of soluble CD4 and a CD4-binding site antibody to immobilized trimers, as detected by surface plasmon resonance. Furthermore, the MPER deletion did not alter the overall three-dimensional structure of the trimers, as viewed by negative-stain electron microscopy. Homogeneous and aggregate-free MPER-truncated SOSIP Env trimers are therefore useful for immunogenicity and structural studies.

Partial enzymatic deglycosylation preserves the structure of cleaved recombinant HIV-1 envelope glycoprotein trimers.

The trimeric envelope glycoprotein complex (Env) is the focus of vaccine development programs aimed at generating protective humoral responses to human immunodeficiency virus type 1 (HIV-1). N-Linked glycans, which constitute almost half of the molecular mass of the external Env domains, produce considerable structural heterogeneity and are a major impediment to crystallization studies. Moreover, by shielding the peptide backbone, glycans can block attempts to generate neutralizing antibodies against a substantial subset of potential epitopes when Env proteins are used as immunogens. Here, we describe the partial deglycosylation of soluble, cleaved recombinant Env trimers by inhibition of the synthesis of complex N-glycans during Env production, followed by treatment with glycosidases under conditions that preserve Env trimer integrity. The partially deglycosylated trimers are stable, and neither abnormally sensitive to proteolytic digestion nor prone to aggregation. Moreover, the deglycosylated trimers retain or increase their ability to bind CD4 and antibodies that are directed to conformational epitopes, including the CD4-binding site and the V3 region. However, as expected, they do not react with glycan-dependent antibodies 2G12 and PGT123, or the C-type lectin receptor DC-SIGN. Electron microscopic analysis shows that partially deglycosylated trimers have a structure similar to fully glycosylated trimers, indicating that removal of glycans does not substantially perturb the structural integrity of the trimer. The glycan-depleted Env trimers should be useful for structural and immunogenicity studies.

Not all sugars are created equal: some mask aversive tastes better than others in an herbivorous insect.

Manduca sexta caterpillars are unusual because they exhibit strong peripheral gustatory responses to sugars, but nevertheless fail to show immediate appetitive responses to them. We hypothesized that the primary function of the peripheral gustatory response to sugars is to mask the taste of noxious compounds, which abound in host plants of M. sexta. We compared 10 s biting responses to water with those to mixtures of a noxious compound [caffeine (Caf) or aristolochic acid (AA)] and various combinations of sugars [i.e. sucrose (Suc), glucose (Glu), inositol (Ino), Suc+Glu, Suc+Ino or Glu+Ino]. The biting assays indicated that the aversive taste of AA was completely masked by Suc+Ino, and partially masked by Suc+Glu, Glu+Ino and Suc, whereas that of Caf was completely masked by Suc+Ino and Suc+Glu, and partially masked by Glu+Ino, Suc and Ino. To examine the contribution of the peripheral taste system to the masking phenomenon, we recorded responses of the maxillary gustatory sensilla to each stimulus mixture. The sugars differed greatly in their capacity to suppress peripheral gustatory responses to AA and Caf: Suc+Ino and Suc+Glu produced the greatest suppression, and Glu and Ino the least. Further, the extent to which each sugar stimulus suppressed the peripheral gustatory responses to AA reliably predicted the extent to which it masked the taste of AA in biting assays; no such predictive relationship was observed for the sugar/Caf mixtures. We conclude that some, but not all, sugars act on both peripheral and central elements of the gustatory system to mask the taste of noxious compounds.