Xiaowei Huang

All Publications

• Cryo-EM structure and molecular mechanism of the jasmonic acid transporter ABCG16 NATURE PLANTS An, N., Huang, X., Yang, Z., Zhang, M., Ma, M., Yu, F., Jing, L., Du, B., Wang, Y., Zhang, X., Zhang, P. 2024

  Abstract

  Jasmonates (JAs) are a class of oxylipin phytohormones including jasmonic acid (JA) and derivatives that regulate plant growth, development and biotic and abiotic stress. A number of transporters have been identified to be responsible for the cellular and subcellular translocation of JAs. However, the mechanistic understanding of how these transporters specifically recognize and transport JAs is scarce. Here we determined the cryogenic electron microscopy structure of JA exporter AtABCG16 in inward-facing apo, JA-bound and occluded conformations, and outward-facing post translocation conformation. AtABCG16 structure forms a homodimer, and each monomer contains a nucleotide-binding domain, a transmembrane domain and an extracellular domain. Structural analyses together with biochemical and plant physiological experiments revealed the molecular mechanism by which AtABCG16 specifically recognizes and transports JA. Structural analyses also revealed that AtABCG16 features a unique bifurcated substrate translocation pathway, which is composed of two independent substrate entrances, two substrate-binding pockets and a shared apoplastic cavity. In addition, residue Phe608 from each monomer is disclosed to function as a gate along the translocation pathway controlling the accessing of substrate JA from the cytoplasm or apoplast. Based on the structural and biochemical analyses, a working model of AtABCG16-mediated JA transport is proposed, which diversifies the molecular mechanisms of ABC transporters.

  View details for DOI 10.1038/s41477-024-01839-0

  View details for Web of Science ID 001347287900001

  View details for PubMedID 39496849

  View details for PubMedCentralID 3662512

• Cryo-EM structure and molecular mechanism of abscisic acid transporter ABCG25 NATURE PLANTS Huang, X., Zhang, X., An, N., Zhang, M., Ma, M., Yang, Y., Jing, L., Wang, Y., Chen, Z., Zhang, P. 2023

  Abstract

  Abscisic acid (ABA) is one of the plant hormones that regulate various physiological processes, including stomatal closure, seed germination and development. ABA is synthesized mainly in vascular tissues and transported to distal sites to exert its physiological functions. Many ABA transporters have been identified, however, the molecular mechanism of ABA transport remains elusive. Here we report the cryogenic electron microscopy structure of the Arabidopsis thaliana adenosine triphosphate-binding cassette G subfamily ABA exporter ABCG25 (AtABCG25) in inward-facing apo conformation, ABA-bound pre-translocation conformation and outward-facing occluded conformation. Structural and biochemical analyses reveal that the ABA bound with ABCG25 adopts a similar configuration as that in ABA receptors and that the ABA-specific binding is dictated by residues from transmembrane helices TM1, TM2 and TM5a of each protomer at the transmembrane domain interface. Comparison of different conformational structures reveals conformational changes, especially those of transmembrane helices and residues constituting the substrate translocation pathway during the cross-membrane transport process. Based on the structural data, a 'gate-flipper' translocation model of ABCG25-mediated ABA cross-membrane transport is proposed. Our structural data on AtABCG25 provide new clues to the physiological study of ABA and shed light on its potential applications in plants and agriculture.

  View details for DOI 10.1038/s41477-023-01509-7

  View details for Web of Science ID 001061915300005

  View details for PubMedID 37666961

  View details for PubMedCentralID 2836657

• GORK K<SUP>+</SUP> channel structure and gating vital to informing stomatal engineering NATURE COMMUNICATIONS Zhang, X., Carroll, W., Nguyen, T., Nguyen, T., Yang, Z., Ma, M., Huang, X., Hills, A., Guo, H., Karnik, R., Blatt, M. R., Zhang, P. 2025; 16 (1): 1961

  Abstract

  The Arabidopsis GORK channel is a major pathway for guard cell K+ efflux that facilitates stomatal closure. GORK is an outwardly-rectifying member of the cyclic-nucleotide binding-homology domain (CNBHD) family of K+ channels with close homologues in all other angiosperms known to date. Its bioengineering has demonstrated the potential for enhanced carbon assimilation and water use efficiency. Here we identify critical domains through structural and functional analysis, highlighting conformations that reflect long-lived closed and pre-open states of GORK. These conformations are marked by interactions at the cytosolic face of the membrane between so-called voltage-sensor, C-linker and CNBHD domains, the latter relocating across 10 Å below the voltage sensor. The interactions center around two coupling sites that functional analysis establish are critical for channel gating. The channel is also subject to putative, ligand-like interactions within the CNBHD, which leads to its gating independence of cyclic nucleotides such as cAMP or cGMP. These findings implicate a multi-step mechanism of semi-independent conformational transitions that underlie channel activity and offer promising new sites for optimizing GORK to engineer stomata.

  View details for DOI 10.1038/s41467-025-57287-7

  View details for Web of Science ID 001432846300008

  View details for PubMedID 40000640

  View details for PubMedCentralID PMC11861651

• Molecular mechanism underlying regulation of Arabidopsis CLCa transporter by nucleotides and phospholipids NATURE COMMUNICATIONS Yang, Z., Zhang, X., Ye, S., Zheng, J., Huang, X., Yu, F., Chen, Z., Cai, S., Zhang, P. 2023; 14 (1): 4879

  Abstract

  Chloride channels (CLCs) transport anion across membrane to regulate ion homeostasis and acidification of intracellular organelles, and are divided into anion channels and anion/proton antiporters. Arabidopsis thaliana CLCa (AtCLCa) transporter localizes to the tonoplast which imports NO3- and to a less extent Cl- from cytoplasm. The activity of AtCLCa and many other CLCs is regulated by nucleotides and phospholipids, however, the molecular mechanism remains unclear. Here we determine the cryo-EM structures of AtCLCa bound with NO3- and Cl-, respectively. Both structures are captured in ATP and PI(4,5)P2 bound conformation. Structural and electrophysiological analyses reveal a previously unidentified N-terminal β-hairpin that is stabilized by ATP binding to block the anion transport pathway, thereby inhibiting the AtCLCa activity. While AMP loses the inhibition capacity due to lack of the β/γ- phosphates required for β-hairpin stabilization. This well explains how AtCLCa senses the ATP/AMP status to regulate the physiological nitrogen-carbon balance. Our data further show that PI(4,5)P2 or PI(3,5)P2 binds to the AtCLCa dimer interface and occupies the proton-exit pathway, which may help to understand the inhibition of AtCLCa by phospholipids to facilitate guard cell vacuole acidification and stomatal closure. In a word, our work suggests the regulatory mechanism of AtCLCa by nucleotides and phospholipids under certain physiological scenarios and provides new insights for future study of CLCs.

  View details for DOI 10.1038/s41467-023-40624-z

  View details for Web of Science ID 001049310000012

  View details for PubMedID 37573431

  View details for PubMedCentralID PMC10423218

• Cryo-EM structure of the CRY2 and CIB1 fragment complex provides insights into CIB1-mediated photosignaling PLANT COMMUNICATIONS Hao, Y., Zhang, X., Liu, Y., Ma, M., Huang, X., Liu, H., Zhang, P. 2023; 4 (2): 100475

  View details for DOI 10.1016/j.xplc.2022.100475

  View details for Web of Science ID 000958522500001

  View details for PubMedID 36371635

  View details for PubMedCentralID PMC10030363

• Constitutive activation of a nuclear-localized calcium channel complex in <i>Medicago truncatula</i> PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Liu, H., Lin, J., Luo, Z., Sun, J., Huang, X., Yang, Y., Xu, J., Wang, Y., Zhang, P., Oldroyd, G. D., Xie, F. 2022; 119 (34): e2205920119

  Abstract

  Nuclear Ca2+ oscillations allow symbiosis signaling, facilitating plant recognition of beneficial microsymbionts, nitrogen-fixing rhizobia, and nutrient-capturing arbuscular mycorrhizal fungi. Two classes of channels, DMI1 and CNGC15, in a complex on the nuclear membrane, coordinate symbiotic Ca2+ oscillations. However, the mechanism of Ca2+ signature generation is unknown. Here, we demonstrate spontaneous activation of this channel complex, through gain-of-function mutations in DMI1, leading to spontaneous nuclear Ca2+ oscillations and spontaneous nodulation, in a CNGC15-dependent manner. The mutations destabilize a hydrogen-bond or salt-bridge network between two RCK domains, with the resultant structural changes, alongside DMI1 cation permeability, activating the channel complex. This channel complex was reconstituted in human HEK293T cell lines, with the resultant calcium influx enhanced by autoactivated DMI1 and CNGC15s. Our results demonstrate the mode of activation of this nuclear channel complex, show that DMI1 and CNGC15 are sufficient to create oscillatory Ca2+ signals, and provide insights into its native mode of induction.

  View details for DOI 10.1073/pnas.2205920119

  View details for Web of Science ID 000972628800005

  View details for PubMedID 35972963

  View details for PubMedCentralID PMC9407390

• Molecular mechanism underlying transport and allosteric inhibition of bicarbonate transporter SbtA PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Fang, S., Huang, X., Zhang, X., Zhang, M., Hao, Y., Guo, H., Liu, L., Yu, F., Zhang, P. 2021; 118 (22)

  Abstract

  SbtA is a high-affinity, sodium-dependent bicarbonate transporter found in the cyanobacterial CO2-concentrating mechanism (CCM). SbtA forms a complex with SbtB, while SbtB allosterically regulates the transport activity of SbtA by binding with adenyl nucleotides. The underlying mechanism of transport and regulation of SbtA is largely unknown. In this study, we report the three-dimensional structures of the cyanobacterial Synechocystis sp. PCC 6803 SbtA-SbtB complex in both the presence and absence of HCO3- and/or AMP at 2.7 Å and 3.2 Å resolution. An analysis of the inward-facing state of the SbtA structure reveals the HCO3-/Na+ binding site, providing evidence for the functional unit as a trimer. A structural comparison found that SbtA adopts an elevator mechanism for bicarbonate transport. A structure-based analysis revealed that the allosteric inhibition of SbtA by SbtB occurs mainly through the T-loop of SbtB, which binds to both the core domain and the scaffold domain of SbtA and locks it in an inward-facing state. T-loop conformation is stabilized by the AMP molecules binding at the SbtB trimer interfaces and may be adjusted by other adenyl nucleotides. The unique regulatory mechanism of SbtA by SbtB makes it important to study inorganic carbon uptake systems in CCM, which can be used to modify photosynthesis in crops.

  View details for DOI 10.1073/pnas.2101632118

  View details for Web of Science ID 000659434200005

  View details for PubMedID 34031249

  View details for PubMedCentralID PMC8179158

• The oligomeric structures of plant cryptochromes NATURE STRUCTURAL & MOLECULAR BIOLOGY Shao, K., Zhang, X., Li, X., Hao, Y., Huang, X., Ma, M., Zhang, M., Yu, F., Liu, H., Zhang, P. 2020; 27 (5): 480-+

  Abstract

  Cryptochromes (CRYs) are a group of evolutionarily conserved flavoproteins found in many organisms. In plants, the well-studied CRY photoreceptor, activated by blue light, plays essential roles in plant growth and development. However, the mechanism of activation remains largely unknown. Here, we determined the oligomeric structures of the blue-light-perceiving PHR domain of Zea mays CRY1 and an Arabidopsis CRY2 constitutively active mutant. The structures form dimers and tetramers whose functional importance is examined in vitro and in vivo with Arabidopsis CRY2. Structure-based analysis suggests that blue light may be perceived by CRY to cause conformational changes, whose precise nature remains to be determined, leading to oligomerization that is essential for downstream signaling. This photoactivation mechanism may be widely used by plant CRYs. Our study reveals a molecular mechanism of plant CRY activation and also paves the way for design of CRY as a more efficient optical switch.

  View details for DOI 10.1038/s41594-020-0420-x

  View details for Web of Science ID 000532487100010

  View details for PubMedID 32398825

  View details for PubMedCentralID 143983

• Structural mechanism of the active bicarbonate transporter from cyanobacteria NATURE PLANTS Wang, C., Sun, B., Zhang, X., Huang, X., Zhang, M., Guo, H., Chen, X., Huang, F., Chen, T., Mi, H., Yu, F., Liu, L., Zhang, P. 2019; 5 (11): 1184-+

  Abstract

  Bicarbonate transporters play essential roles in pH homeostasis in mammals and photosynthesis in aquatic photoautotrophs. A number of bicarbonate transporters have been characterized, among which is BicA-a low-affinity, high-flux SLC26-family bicarbonate transporter involved in cyanobacterial CO2-concentrating mechanisms (CCMs) that accumulate CO2 and improve photosynthetic carbon fixation. Here, we report the three-dimensional structure of BicA from Synechocystis sp. PCC6803. Crystal structures of the transmembrane domain (BicATM) and the cytoplasmic STAS domain (BicASTAS) of BicA were solved. BicATM was captured in an inward-facing HCO3--bound conformation and adopts a '7+7' fold monomer. HCO3- binds to a cytoplasm-facing hydrophilic pocket within the membrane. BicASTAS is assembled as a compact homodimer structure and is required for the dimerization of BicA. The dimeric structure of BicA was further analysed using cryo-electron microscopy and physiological analysis of the full-length BicA, and may represent the physiological unit of SLC26-family transporters. Comparing the BicATM structure with the outward-facing transmembrane domain structures of other bicarbonate transporters suggests an elevator transport mechanism that is applicable to the SLC26/4 family of sodium-dependent bicarbonate transporters. This study advances our knowledge of the structures and functions of cyanobacterial bicarbonate transporters, and will inform strategies for bioengineering functional BicA in heterologous organisms to increase assimilation of CO2.

  View details for DOI 10.1038/s41477-019-0538-1

  View details for Web of Science ID 000496526100018

  View details for PubMedID 31712753

  View details for PubMedCentralID 5616186