The major facilitator superfamily glucose transporters, exemplified by human GLUT1-4, have been central to the study of solute transport. Using lipidic cubic phase crystallization and microfocus X-ray diffraction, we determined the structure of human GLUT3 in complex with D-glucose at 1.5 Å resolution in an outward-occluded conformation. The high-resolution structure allows discrimination of both α- and β-anomers of D-glucose. Two additional structures of GLUT3 bound to the exofacial inhibitor maltose were obtained at 2.6 Å in the outward-open and 2.4 Å in the outward-occluded states. In all three structures, the ligands are predominantly coordinated by polar residues from the carboxy terminal domain. Conformational transition from outward-open to outward-occluded entails a prominent local rearrangement of the extracellular part of transmembrane segment TM7. Comparison of the outward-facing GLUT3 structures with the inward-open GLUT1 provides insights into the alternating access cycle for GLUTs, whereby the C-terminal domain provides the primary substrate-binding site and the amino-terminal domain undergoes rigid-body rotation with respect to the C-terminal domain. Our studies provide an important framework for the mechanistic and kinetic understanding of GLUTs and shed light on structure-guided ligand design.

The interactions between phytohormones are crucial for plants to adapt to complex environmental changes. One example is the ethylene-regulated local auxin biosynthesis in roots, which partly contributes to ethylene-directed root development and gravitropism. Using a chemical biology approach, we identified a small molecule, L-kynurenine (Kyn), which effectively inhibited ethylene responses in Arabidopsis thaliana root tissues. Kyn application repressed nuclear accumulation of the ETHYLENE INSENSITIVE3 (EIN3) transcription factor. Moreover, Kyn application decreased ethylene-induced auxin biosynthesis in roots, and TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1/TRYPTOPHAN AMINOTRANSFERASE RELATEDs (TAA1/TARs), the key enzymes in the indole-3-pyruvic acid pathway of auxin biosynthesis, were identified as the molecular targets of Kyn. Further biochemical and phenotypic analyses revealed that Kyn, being an alternate substrate, competitively inhibits TAA1/TAR activity, and Kyn treatment mimicked the loss of TAA1/TAR functions. Molecular modeling and sequence alignments suggested that Kyn effectively and selectively binds to the substrate pocket of TAA1/TAR proteins but not those of other families of aminotransferases. To elucidate the destabilizing effect of Kyn on EIN3, we further found that auxin enhanced EIN3 nuclear accumulation in an EIN3 BINDING F-BOX PROTEIN1 (EBF1)/EBF2-dependent manner, suggesting the existence of a positive feedback loop between auxin biosynthesis and ethylene signaling. Thus, our study not only reveals a new level of interactions between ethylene and auxin pathways but also offers an efficient method to explore and exploit TAA1/TAR-dependent auxin biosynthesis.

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FocA belongs to the formate-nitrate transporter family and plays an essential role in the export and uptake of formate in organisms. According to the available crystal structures, the N-terminal residues of FocA are structurally featureless at physiological conditions but at reduced pH form helices to harbor the cytoplasmic entrance of the substrate permeation pathway, which apparently explains the cessation of electrical signal observed in electrophysiological experiments. In this work, we found by structural analysis and molecular dynamics simulations that those N-terminal helices cannot effectively preclude the substrate permeation. Equilibrium simulations and thermodynamic calculations suggest that FocA is permeable to both formate and formic acid, the latter of which is transparent to electrophysiological studies as an electrically neutral species. Hence, the cease of electrical current at acidic pH may be caused by the change of the transported substrate from formate to formic acid. In addition, the mechanism of formate export at physiological pH is discussed.

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The lactose permease of Escherichia coli (LacY), a dynamic polytopic membrane protein, catalyzes galactoside-H + symport and operates by an alternating access mechanism that exhibits multiple conformations, the distribution of which is altered by sugar binding. We have developed single-domain camelid nanobodies (Nbs) against a mutant in an outward (periplasmic)-open conformation to stabilize this state of the protein. Here we describe an X-ray crystal structure of a complex between a double-Trp mutant (Gly46→Trp/Gly262→Trp) and an Nb in which free access to the sugar-binding site from the periplasmic cavity is observed. The structure confirms biochemical data indicating that the Nb binds stoichiometrically with nanomolar affinity to the periplasmic face of LacY primarily to the C-terminal six-helix bundle. The structure is novel because the pathway to the sugar-binding site is constricted and the central cavity containing the galactoside-binding site is empty. Although Phe27 narrows the periplasmic cavity, sugar is freely accessible to the binding site. Remarkably, the side chains directly involved in binding galactosides remain in the same position in the absence or presence of bound sugar.X-ray structure | bioenergetics | membrane transporter | fluorescence | kinetics T he lactose permease of Escherichia coli (LacY) catalyzes the coupled transport of a galactopyranoside and an H + (galactoside-H + symport) across the cytoplasmic membrane (reviewed in refs. 1-3). Therefore, in the presence of an H + electrochemical gradient (ΔμH+; interior negative and/or alkaline), galactopyranosides can be concentrated 50-to 100-fold over the external concentration (4, 5). However, due to the activity of β-galactosidase, no free lactose is found within the cell under physiological conditions. So why does E. coli need an active transport system for lactose? The answer lies in the kinetics, as the major effect of ΔμH+ is to decrease the K m for transport (6, 7). By increasing the kinetic efficiency of LacY, ΔμH+ enables the cell to assimilate lactose effectively even when the environmental concentration is low.Initial X-ray crystal structures of conformationally restricted LacY (8-10), as well as WT LacY (11) + and is the poster child for the major facilitator superfamily, the largest family of membrane transport proteins. A detailed mechanism has been postulated involving alternating access of sugar-and H + -binding sites to either side of the membrane that is driven by sugar binding and dissociation and independent of the H + electrochemical gradient, which acts kinetically. To characterize structural intermediates in the transport cycle, stable conformers are essential, and camelid single-domain nanobodies (Nbs) are particularly useful in this context. Described herein is a structure of a LacY-Nb complex in which access to the sugar-binding site from the periplasmic cavity is diffusion-limited.Author contributions: X.J., I.S., V.K., N.Y., and H.R.K. designed research; X.J., I.S., V.K., J.W., K.H., and M.K. performed research...

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Raising public awareness of the Sustainable Development Goals (SDGs) is a critical prerequisite for their implementation. However, little is known about attitude formation among the public toward SDGs at the national level. We explored this topic in China, a country that has emerged as a leading world economy with strong transformational imperatives to work toward sustainable development. Following Chaiken’s heuristic–systematic model and using data from an online survey with 4128 valid respondents, this study investigated the factors that affect public support for SDGs and explains how individuals form supportive attitudes. Our empirical evidence showed that in China, first, public support is mainly shaped by demographic attributes (gender, age, and educational attainment), value predispositions (e.g., altruistic values and anthropocentric worldviews), and the level of SDG-relevant knowledge. Second, an interaction effect exists between value predispositions and knowledge among the public concerning support for SDGs. Third, the Chinese public views the implementation of SDGs as a part of development policy rather than environmental policy. This study provides empirical findings on the factors that account for public attitudes toward SDGs, outlining some useful implications for designing policy tools that would bolster SDG action.