Xinqi Gong

Crystal structure of a bacterial homologue of glucose transporters GLUT1-4.

Glucose transporters are essential for metabolism of glucose in cells of diverse organisms from microbes to humans, exemplified by the disease-related human proteins GLUT1, 2, 3 and 4. Despite rigorous efforts, the structural information for GLUT1–4 or their homologues remains largely unknown. Here we report three related crystal structures of XylE, an Escherichia coli homologue of GLUT1–4, in complex with d-xylose, d-glucose and 6-bromo-6-deoxy-d-glucose, at resolutions of 2.8, 2.9 and 2.6 Å, respectively. The structure consists of a typical major facilitator superfamily fold of 12 transmembrane segments and a unique intracellular four-helix domain. XylE was captured in an outward-facing, partly occluded conformation. Most of the important amino acids responsible for recognition of d-xylose or d-glucose are invariant in GLUT1–4, suggesting functional and mechanistic conservations. Structure-based modelling of GLUT1–4 allows mapping and interpretation of disease-related mutations. The structural and biochemical information reported here constitutes an important framework for mechanistic understanding of glucose transporters and sugar porters in general.

A Small-Molecule Screen Identifies l-Kynurenine as a Competitive Inhibitor of TAA1/TAR Activity in Ethylene-Directed Auxin Biosynthesis and Root Growth in Arabidopsis

In this work, Kyn is identified as an auxin biosynthesis inhibitor that effectively and selectively targets TAA1-like Trp aminotransferases. Moreover, it describes a previously undiscovered positive feedback loop between auxin biosynthesis and ethylene signaling pathways in roots. 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.

Structure of a presenilin family intramembrane aspartate protease

Presenilin and signal peptide peptidase (SPP) are intramembrane aspartyl proteases that regulate important biological functions in eukaryotes. Mechanistic understanding of presenilin and SPP has been hampered by lack of relevant structural information. Here we report the crystal structure of a presenilin/SPP homologue (PSH) from the archaeon Methanoculleus marisnigri JR1. The protease, comprising nine transmembrane segments (TMs), adopts a previously unreported protein fold. The amino-terminal domain, consisting of TM1–6, forms a horseshoe-shaped structure, surrounding TM7–9 of the carboxy-terminal domain. The two catalytic aspartate residues are located on the cytoplasmic side of TM6 and TM7, spatially close to each other and approximately 8 Å into the lipid membrane surface. Water molecules gain constant access to the catalytic aspartates through a large cavity between the amino- and carboxy-terminal domains. Structural analysis reveals insights into the presenilin/SPP family of intramembrane proteases.

Structure and Mechanism of a Nitrate Transporter

The nitrate/nitrite transporters NarK and NarU play an important role in nitrogen homeostasis in bacteria and belong to the nitrate/nitrite porter family (NNP) of the major facilitator superfamily (MFS) fold. The structure and functional mechanism of NarK and NarU remain unknown. Here, we report the crystal structure of NarU at a resolution of 3.1 Å and systematic biochemical characterization. The two molecules of NarU in an asymmetric unit exhibit two distinct conformational states: occluded and partially inward-open. The substrate molecule nitrate appears to be coordinated by four highly conserved, charged, or polar amino acids. Structural and biochemical analyses allowed the identification of key amino acids that are involved in substrate gating and transport. The observed conformational differences of NarU, together with unique sequence features of the NNP family transporters, suggest a transport mechanism that might deviate from the canonical rocker-switch model.

Structure of a bacterial energy-coupling factor transporter

The energy-coupling factor (ECF) transporters constitute a novel family of conserved membrane transporters in prokaryotes that have a similar domain organization to the ATP-binding cassette transporters. Each ECF transporter comprises a pair of cytosolic ATPases (the A and A′ components, or EcfA and EcfA′), a membrane-embedded substrate-binding protein (the S component, or EcfS) and a transmembrane energy-coupling component (the T component, or EcfT) that links the EcfA–EcfA′ subcomplex to EcfS. The structure and transport mechanism of the quaternary ECF transporter remain largely unknown. Here we report the crystal structure of a nucleotide-free ECF transporter from Lactobacillus brevis at a resolution of 3.5 Å. The T component has a horseshoe-shaped open architecture, with five α-helices as transmembrane segments and two cytoplasmic α-helices as coupling modules connecting to the A and A′ components. Strikingly, the S component, thought to be specific for hydroxymethyl pyrimidine, lies horizontally along the lipid membrane and is bound exclusively by the five transmembrane segments and the two cytoplasmic helices of the T component. These structural features suggest a plausible working model for the transport cycle of the ECF transporters.

Molecular Mechanism for Inhibition of a Critical Component in the Arabidopsis thaliana Abscisic Acid Signal Transduction Pathways, SnRK2.6, by Protein Phosphatase ABI1

Background: The antagonistic complex of SnRK2.6 and ABI1 regulates abscisic acid (ABA) signaling in plants. Results: Presented here are the structure of SnRK2.6 kinase domain, and biochemical and computational characterizations of the ABI1-SnRK2.6 complex. Conclusion: Our studies revealed the molecular basis for ABI1-mediated inhibition of SnRK2.6. Significance: The studies advanced our understanding of the downstream signal transduction of ABA through SnRK2s and protein phosphatase type 2Cs. Subclass III SnRK2s (SnRK2.6/2.3/2.2) are the key positive regulators of abscisic acid (ABA) signal transduction in Arabidopsis thaliana. The kinases, activated by ABA or osmotic stress, phosphorylate stress-related transcription factors and ion channels, which ultimately leads to the protection of plants from dehydration or high salinity. In the absence of stressors, SnRK2s are subject to negative regulation by group A protein phosphatase type 2Cs (PP2C), whereas the underlying molecular mechanism remains to be elucidated. Here we report the crystal structure of the kinase domain of SnRK2.6 at 2.6-Å resolution. Structure-guided biochemical analyses identified two distinct interfaces between SnRK2.6 and ABI1, a member of group A PP2Cs. Structural modeling suggested that the two interfaces lock SnRK2.6 and ABI1 in an orientation such that the activation loop of SnRK2.6 is posited to the catalytic site of ABI1 for dephosphorylation. These studies revealed the molecular basis for PP2Cs-mediated inhibition of SnRK2s and provided important insights into the downstream signal transduction of ABA.

Structure and mechanism of a eukaryotic transmembrane ascorbate-dependent oxidoreductase

Significance Vitamin C (also known as ascorbate), an essential nutrient for humans, plays an important role in protection against oxidative stress. The ascorbate-dependent oxidoreductase cytochrome b561 (Cyt b561) is a family of highly conserved, multipass transmembrane enzymes found only in eukaryotes. Cyt b561 plays a key role in ascorbate recycling and many other important physiological processes, such as iron absorption. The atomic structure and functional mechanism of Cyt b561 remain unknown. In this study, we report the high-resolution crystal structures of Cyt b561 in both ascorbate-free and ascorbate-bound states. Our structural and biochemical analyses identify a general functional mechanism for the Cyt b561 family. Vitamin C, also known as ascorbate, is required in numerous essential metabolic reactions in eukaryotes. The eukaryotic ascorbate-dependent oxidoreductase cytochrome b561 (Cyt b561), a family of highly conserved transmembrane enzymes, plays an important role in ascorbate recycling and iron absorption. Although Cyt b561 was identified four decades ago, its atomic structure and functional mechanism remain largely unknown. Here, we report the high-resolution crystal structures of cytochrome b561 from Arabidopsis thaliana in both substrate-free and substrate-bound states. Cyt b561 forms a homodimer, with each protomer consisting of six transmembrane helices and two heme groups. The negatively charged substrate ascorbate, or monodehydroascorbate, is enclosed in a positively charged pocket on either side of the membrane. Two highly conserved amino acids, Lys81 and His106, play an essential role in substrate recognition and catalysis. Our structural and biochemical analyses allow the proposition of a general electron transfer mechanism for members of the Cyt b561 family.