I03-Macromolecular Crystallography
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Mauricio P.
Contreras
,
Hsuan
Pai
,
Muniyandi
Selvaraj
,
Amirali
Toghani
,
David M.
Lawson
,
Yasin
Tumtas
,
Cian
Duggan
,
Enoch Lok Him
Yuen
,
Clare E. M.
Stevenson
,
Adeline
Harant
,
Abbas
Maqbool
,
Chih-Hang
Wu
,
Tolga O.
Bozkurt
,
Sophien
Kamoun
,
Lida
Derevnina
Diamond Proposal Number(s):
[18565]
Open Access
Abstract: Parasites counteract host immunity by suppressing helper nucleotide binding and leucine-rich repeat (NLR) proteins that function as central nodes in immune receptor networks. Understanding the mechanisms of immunosuppression can lead to strategies for bioengineering disease resistance. Here, we show that a cyst nematode virulence effector binds and inhibits oligomerization of the helper NLR protein NRC2 by physically preventing intramolecular rearrangements required for activation. An amino acid polymorphism at the binding interface between NRC2 and the inhibitor is sufficient for this helper NLR to evade immune suppression, thereby restoring the activity of multiple disease resistance genes. This points to a potential strategy for resurrecting disease resistance in crop genomes.
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May 2023
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[30015, 23316, 17844]
Open Access
Abstract: Sucrose import from photosynthetic tissues into the phloem is mediated by transporters from the low-affinity sucrose transporter family (SUC/SUT family). Furthermore, sucrose redistribution to other tissues is driven by phloem sap movement, the product of high turgor pressure created by this import activity. Additionally, sink organs such as fruits, cereals and seeds that accumulate high concentrations of sugar also depend on this active transport of sucrose. Here we present the structure of the sucrose–proton symporter, Arabidopsis thaliana SUC1, in an outward open conformation at 2.7 Å resolution, together with molecular dynamics simulations and biochemical characterization. We identify the key acidic residue required for proton-driven sucrose uptake and describe how protonation and sucrose binding are strongly coupled. Sucrose binding is a two-step process, with initial recognition mediated by the glucosyl moiety binding directly to the key acidic residue in a stringent pH-dependent manner. Our results explain how low-affinity sucrose transport is achieved in plants, and pinpoint a range of SUC binders that help define selectivity. Our data demonstrate a new mode for proton-driven symport with links to cation-driven symport and provide a broad model for general low-affinity transport in highly enriched substrate environments.
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May 2023
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I03-Macromolecular Crystallography
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Simon
Stael
,
Igor
Sabljic
,
Dominique
Audenaert
,
Thilde
Andersson
,
Liana
Tsiatsiani
,
Robert P.
Kumpf
,
Andreu
Vidal-Albalat
,
Cecilia
Lindgren
,
Dominique
Vercammen
,
Silke
Jacques
,
Long
Nguyen
,
Maria
Njo
,
Álvaro D.
Fernández-Fernández
,
Tine
Beunens
,
Evy
Timmerman
,
Kris
Gevaert
,
Marc
Van Montagu
,
Jerry
Stahlberg
,
Peter V.
Bozhkov
,
Anna
Linusson
,
Tom
Beeckman
,
Frank
Van Breusegem
Diamond Proposal Number(s):
[23773]
Abstract: Metacaspases are part of an evolutionarily broad family of multifunctional cysteine proteases, involved in disease and normal development. As the structure–function relationship of metacaspases remains poorly understood, we solved the X-ray crystal structure of an Arabidopsis thaliana type II metacaspase (AtMCA-IIf) belonging to a particular subgroup not requiring calcium ions for activation. To study metacaspase activity in plants, we developed an in vitro chemical screen to identify small molecule metacaspase inhibitors and found several hits with a minimal thioxodihydropyrimidine-dione structure, of which some are specific AtMCA-IIf inhibitors. We provide mechanistic insight into the basis of inhibition by the TDP-containing compounds through molecular docking onto the AtMCA-IIf crystal structure. Finally, a TDP-containing compound (TDP6) effectively hampered lateral root emergence in vivo, probably through inhibition of metacaspases specifically expressed in the endodermal cells overlying developing lateral root primordia. In the future, the small compound inhibitors and crystal structure of AtMCA-IIf can be used to study metacaspases in other species, such as important human pathogens, including those causing neglected diseases.
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May 2023
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Josephine H. R.
Maidment
,
Motoki
Shimizu
,
Adam R.
Bentham
,
Sham
Vera
,
Marina
Franceschetti
,
Apinya
Longya
,
Clare E. M.
Stevenson
,
Juan Carlos
De La Concepcion
,
Aleksandra
Bialas
,
Sophien
Kamoun
,
Ryohei
Terauchi
,
Mark J.
Banfield
Diamond Proposal Number(s):
[13467, 18565]
Open Access
Abstract: A subset of plant intracellular NLR immune receptors detect effector proteins, secreted by phytopathogens to promote infection, through unconventional integrated domains which resemble the effector’s host targets. Direct binding of effectors to these integrated domains activates plant defenses. The rice NLR receptor Pik-1 binds the Magnaporthe oryzae effector AVR-Pik through an integrated heavy metal-associated (HMA) domain. However, the stealthy alleles AVR-PikC and AVR-PikF avoid interaction with Pik-HMA and evade host defenses. Here, we exploited knowledge of the biochemical interactions between AVR-Pik and its host target, OsHIPP19, to engineer novel Pik-1 variants that respond to AVR-PikC/F. First, we exchanged the HMA domain of Pikp-1 for OsHIPP19-HMA, demonstrating that effector targets can be incorporated into NLR receptors to provide novel recognition profiles. Second, we used the structure of OsHIPP19-HMA to guide the mutagenesis of Pikp-HMA to expand its recognition profile. We demonstrate that the extended recognition profiles of engineered Pikp-1 variants correlate with effector binding in planta and in vitro, and with the gain of new contacts across the effector/HMA interface. Crucially, transgenic rice producing the engineered Pikp-1 variants was resistant to blast fungus isolates carrying AVR-PikC or AVR-PikF. These results demonstrate that effector target-guided engineering of NLR receptors can provide new-to-nature disease resistance in crops.
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May 2023
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Open Access
Abstract: The arbuscular mycorrhizal (AM) symbiosis is an ancient and highly conserved mutualism between plant and fungal symbionts, in which a highly specialized membrane-delimited fungal arbuscule acts as the symbiotic interface for nutrient exchange and signaling. As a ubiquitous means of biomolecule transport and intercellular communication, extracellular vesicles (EVs) are likely to play a role in this intimate cross-kingdom symbiosis, yet, there is a lack of research investigating the importance of EVs in AM symbiosis despite known roles in microbial interactions in both animal and plant pathosystems. Clarifying the current understanding of EVs in this symbiosis in light of recent ultrastructural observations is paramount to guiding future investigations in the field, and, to this end, this review summarizes recent research investigating these areas. Namely, this review discusses the available knowledge regarding biogenesis pathways and marker proteins associated with the various plant EV subclasses, EV trafficking pathways during symbiosis, and the endocytic mechanisms implicated in the uptake of these EVs.
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Apr 2023
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18565]
Open Access
Abstract: Myo-inositol tris/tetrakisphosphate kinases (ITPKs) catalyze diverse phosphotransfer reactions with myo-inositol phosphate and myo-inositol pyrophosphate substrates. However, the lack of structures of nucleotide-coordinated plant ITPKs thwarts a rational understanding of phosphotransfer reactions of the family. Arabidopsis possesses a family of four ITPKs of which two isoforms, ITPK1 and ITPK4, control inositol hexakisphosphate and inositol pyrophosphate levels directly or by provision of precursors. Here, we describe the specificity of Arabidopsis ITPK4 to pairs of enantiomers of diverse inositol polyphosphates and show how substrate specificity differs from Arabidopsis ITPK1. Moreover, we provide a description of the crystal structure of ATP-coordinated AtITPK4 at 2.11 Å resolution that along with description of the enantiospecificity of the enzyme affords a molecular explanation for the diverse phosphotransferase activity of this enzyme. That Arabidopsis ITPK4 has a Km for ATP in the tens of micromolar range, potentially explains how, despite the large-scale abolition of InsP6, InsP7 and InsP8 synthesis in Atitpk4 mutants, Atitpk4 lacks the phosphate starvation responses of Atitpk1 mutants. We further demonstrate that Arabidopsis ITPK4 and its homologs in other plants possess an N-terminal haloacid dehalogenase-like fold not previously described. The structural and enzymological information revealed will guide elucidation of ITPK4 function in diverse physiological contexts, including InsP8-dependent aspects of plant biology.
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Mar 2023
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[21426]
Open Access
Abstract: Transcriptional silencing through the Polycomb silencing machinery utilizes a “read-write” mechanism involving histone tail modifications. However, nucleation of silencing and long-term stable transmission of the silenced state also requires P-olycomb Repressive Complex 2 (PRC2) accessory proteins, whose molecular role is poorly understood. The Arabidopsis VEL proteins are accessory proteins that interact with PRC2 to nucleate and propagate silencing at the FLOWERING LOCUS C (FLC) locus, enabling early flowering in spring. Here, we report that VEL proteins contain a domain related to an atypical four-helix bundle that engages in spontaneous concentration-dependent head-to-tail polymerization to assemble dynamic biomolecular condensates. Mutations blocking polymerization of this VEL domain prevent Polycomb silencing at FLC. Plant VEL proteins thus facilitate assembly of dynamic multivalent Polycomb complexes required for inheritance of the silenced state.
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Nov 2022
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18565]
Open Access
Abstract: Exocytosis plays an important role in plant–microbe interactions, in both pathogenesis and symbiosis. Exo70 proteins are integral components of the exocyst, an octameric complex that mediates tethering of vesicles to membranes in eukaryotes. Although plant Exo70s are known to be targeted by pathogen effectors, the underpinning molecular mechanisms and the impact of this interaction on infection are poorly understood. Here, we show the molecular basis of the association between the effector AVR-Pii of the blast fungus Maganaporthe oryzae and rice Exo70 alleles OsExo70F2 and OsExo70F3, which is sensed by the immune receptor pair Pii via an integrated RIN4/NOI domain. The crystal structure of AVR-Pii in complex with OsExo70F2 reveals that the effector binds to a conserved hydrophobic pocket in Exo70, defining an effector/target binding interface. Structure-guided and random mutagenesis validates the importance of AVR-Pii residues at the Exo70 binding interface to sustain protein association and disease resistance in rice when challenged with fungal strains expressing effector mutants. Furthermore, the structure of AVR-Pii defines a zinc-finger effector fold (ZiF) distinct from the MAX (Magnaporthe Avrs and ToxB-like) fold previously described for a majority of characterized M. oryzae effectors. Our data suggest that blast fungus ZiF effectors bind a conserved Exo70 interface to manipulate plant exocytosis and that these effectors are also baited by plant immune receptors, pointing to new opportunities for engineering disease resistance.
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Oct 2022
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13467]
Open Access
Abstract: Medium-chain alcohol dehydrogenases (ADHs) comprise a highly conserved enzyme family that catalyse the reversible reduction of aldehydes. However, recent discoveries in plant natural product biosynthesis suggest that the catalytic repertoire of ADHs has been expanded. Here we report the crystal structure of dihydroprecondylocarpine acetate synthase (DPAS), an ADH that catalyses the non-canonical 1,4-reduction of an α,β -unsaturated iminium moiety. Comparison with structures of plant-derived ADHs suggest the 1,4-iminium reduction does not require a proton relay or the presence of a catalytic zinc ion in contrast to canonical 1,2-aldehyde reducing ADHs that require the catalytic zinc and a proton relay. Furthermore, ADHs that catalysed 1,2-iminium reduction required the presence of the catalytic zinc and the loss of the proton relay. This suggests how the ADH active site can be modified to perform atypical carbonyl reductions, providing insight into how chemical reactions are diversified in plant metabolism.
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Oct 2022
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Krios II-Titan Krios II at Diamond
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Diamond Proposal Number(s):
[21404, 27980]
Open Access
Abstract: Auxins are hormones that have central roles and control nearly all aspects of growth and development in plants1,2,3. The proteins in the PIN-FORMED (PIN) family (also known as the auxin efflux carrier family) are key participants in this process and control auxin export from the cytosol to the extracellular space4,5,6,7,8,9. Owing to a lack of structural and biochemical data, the molecular mechanism of PIN-mediated auxin transport is not understood. Here we present biophysical analysis together with three structures of Arabidopsis thaliana PIN8: two outward-facing conformations with and without auxin, and one inward-facing conformation bound to the herbicide naphthylphthalamic acid. The structure forms a homodimer, with each monomer divided into a transport and scaffold domain with a clearly defined auxin binding site. Next to the binding site, a proline–proline crossover is a pivot point for structural changes associated with transport, which we show to be independent of proton and ion gradients and probably driven by the negative charge of the auxin. The structures and biochemical data reveal an elevator-type transport mechanism reminiscent of bile acid/sodium symporters, bicarbonate/sodium symporters and sodium/proton antiporters. Our results provide a comprehensive molecular model for auxin recognition and transport by PINs, link and expand on a well-known conceptual framework for transport, and explain a central mechanism of polar auxin transport, a core feature of plant physiology, growth and development.
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Sep 2022
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