I03-Macromolecular Crystallography
|
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.
|
May 2023
|
|
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
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.
|
May 2023
|
|
I04-Macromolecular Crystallography
|
Martina
Durcik
,
Andrej Emanuel
Cotman
,
Žan
Toplak
,
Štefan
Možina
,
Žiga
Skok
,
Petra Eva
Szili
,
Márton
Czikkely
,
Elvin
Maharramov
,
Thu Hien
Vu
,
Maria Vittoria
Piras
,
Nace
Zidar
,
Janez
Ilaš
,
Anamarija
Zega
,
Jurij
Trontelj
,
Luis A.
Pardo
,
Diarmaid
Hughes
,
Douglas
Huseby
,
Tália
Berruga-Fernández
,
Sha
Cao
,
Ivailo
Simoff
,
Richard
Svensson
,
Sergiy V.
Korol
,
Zhe
Jin
,
Francisca
Vicente
,
Maria C.
Ramos
,
Julia E. A.
Mundy
,
Anthony
Maxwell
,
Clare E. M.
Stevenson
,
David M.
Lawson
,
Björn
Glinghammar
,
Eva
Sjöström
,
Martin
Bohlin
,
Joanna
Oreskär
,
Sofie
Alvér
,
Guido V.
Janssen
,
Geert Jan
Sterk
,
Danijel
Kikelj
,
Csaba
Pal
,
Tihomir
Tomašič
,
Lucija
Peterlin Mašič
Diamond Proposal Number(s):
[25108]
Open Access
Abstract: A new series of dual low nanomolar benzothiazole inhibitors of bacterial DNA gyrase and topoisomerase IV were developed. The resulting compounds show excellent broad-spectrum antibacterial activities against Gram-positive Enterococcus faecalis, Enterococcus faecium and multidrug resistant (MDR) Staphylococcus aureus strains [best compound minimal inhibitory concentrations (MICs): range, <0.03125–0.25 μg/mL] and against the Gram-negatives Acinetobacter baumannii and Klebsiella pneumoniae (best compound MICs: range, 1–4 μg/mL). Lead compound 7a was identified with favorable solubility and plasma protein binding, good metabolic stability, selectivity for bacterial topoisomerases, and no toxicity issues. The crystal structure of 7a in complex with Pseudomonas aeruginosa GyrB24 revealed its binding mode at the ATP-binding site. Expanded profiling of 7a and 7h showed potent antibacterial activity against over 100 MDR and non-MDR strains of A. baumannii and several other Gram-positive and Gram-negative strains. Ultimately, in vivo efficacy of 7a in a mouse model of vancomycin-intermediate S. aureus thigh infection was also demonstrated.
|
Mar 2023
|
|
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Andrej Emanuel
Cotman
,
Martina
Durcik
,
Davide
Benedetto Tiz
,
Federica
Fulgheri
,
Daniela
Secci
,
Maša
Sterle
,
Štefan
Možina
,
Žiga
Skok
,
Nace
Zidar
,
Anamarija
Zega
,
Janez
Ilaš
,
Lucija
Peterlin Mašič
,
Tihomir
Tomašič
,
Diarmaid
Hughes
,
Douglas L.
Huseby
,
Sha
Cao
,
Linnéa
Garoff
,
Talía
Berruga Fernández
,
Paraskevi
Giachou
,
Lisa
Crone
,
Ivailo
Simoff
,
Richard
Svensson
,
Bryndis
Birnir
,
Sergiy V.
Korol
,
Zhe
Jin
,
Francisca
Vicente
,
Maria C.
Ramos
,
Mercedes
De La Cruz
,
Björn
Glinghammar
,
Lena
Lenhammar
,
Sara R.
Henderson
,
Julia E. A.
Mundy
,
Anthony
Maxwell
,
Claren E. M.
Stevenson
,
David M.
Lawson
,
Guido V.
Janssen
,
Geert Jan
Sterk
,
Danijel
Kikelj
Diamond Proposal Number(s):
[18565, 25108]
Open Access
Abstract: We have developed compounds with a promising activity against Acinetobacter baumannii and Pseudomonas aeruginosa, which are both on the WHO priority list of antibiotic-resistant bacteria. Starting from DNA gyrase inhibitor 1, we identified compound 27, featuring a 10-fold improved aqueous solubility, a 10-fold improved inhibition of topoisomerase IV from A. baumannii and P. aeruginosa, a 10-fold decreased inhibition of human topoisomerase IIα, and no cross-resistance to novobiocin. Cocrystal structures of 1 in complex with Escherichia coli GyrB24 and (S)-27 in complex with A. baumannii GyrB23 and P. aeruginosa GyrB24 revealed their binding to the ATP-binding pocket of the GyrB subunit. In further optimization steps, solubility, plasma free fraction, and other ADME properties of 27 were improved by fine-tuning of lipophilicity. In particular, analogs of 27 with retained anti-Gram-negative activity and improved plasma free fraction were identified. The series was found to be nongenotoxic, nonmutagenic, devoid of mitochondrial toxicity, and possessed no ion channel liabilities.
|
Jan 2023
|
|
I03-Macromolecular Crystallography
|
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.
|
Oct 2022
|
|
I03-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[13467]
Abstract: Bacteria are equipped with a diverse set of regulatory tools that allow them to quickly adapt to their environment. The RimK system allows for Pseudomonas spp. to adapt through post-transcriptional regulation by altering the ribosomal subunit RpsF. RimK is found in a wide range of bacteria with a conserved amino acid sequence, however the genetic context and the role of this protein is highly diverse. By solving and comparing the structures of RimK homologues from two related but functionally divergent systems, we uncovered key structural differences that likely contribute to the different activity levels of each of these homologues. Moreover, we were able to clearly resolve the active site of this protein for the first time, resolving binding of the glutamate substrate. This work advances our understanding of how subtle differences in protein sequence and structure can have profound effects on protein activity, which can in turn result in widespread mechanistic changes.
|
Sep 2022
|
|
I03-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[18565]
Open Access
Abstract: Plants use intracellular nucleotide-binding domain (NBD) and leucine-rich repeat (LRR)–containing immune receptors (NLRs) to detect pathogen-derived effector proteins. The Arabidopsis NLR pair RRS1-R/RPS4 confers disease resistance to different bacterial pathogens by perceiving the structurally distinct effectors AvrRps4 from Pseudomonas syringae pv. pisi and PopP2 from Ralstonia solanacearum via an integrated WRKY domain in RRS1-R. How the WRKY domain of RRS1 (RRS1WRKY) perceives distinct classes of effector to initiate an immune response is unknown. Here, we report the crystal structure of the in planta processed C-terminal domain of AvrRps4 (AvrRps4C) in complex with RRS1WRKY. Perception of AvrRps4C by RRS1WRKY is mediated by the β2-β3 segment of RRS1WRKY that binds an electronegative patch on the surface of AvrRps4C. Structure-based mutations that disrupt AvrRps4C–RRS1WRKY interactions in vitro compromise RRS1/RPS4-dependent immune responses. We also show that AvrRps4C can associate with the WRKY domain of the related but distinct RRS1B/RPS4B NLR pair, and the DNA-binding domain of AtWRKY41, with similar binding affinities and how effector binding interferes with WRKY–W-box DNA interactions. This work demonstrates how integrated domains in plant NLRs can directly bind structurally distinct effectors to initiate immunity.
|
Dec 2021
|
|
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
|
Diamond Proposal Number(s):
[13467, 18565]
Open Access
Abstract: Proper chromosome segregation is essential in all living organisms. The ParA-ParB-parS system is widely employed for chromosome segregation in bacteria. Previously, we showed that Caulobacter crescentus ParB requires cytidine triphosphate to escape the nucleation site parS and spread by sliding to the neighboring DNA (Jalal et al., 2020). Here, we provide the structural basis for this transition from nucleation to spreading by solving co-crystal structures of a C-terminal domain truncated C. crescentus ParB with parS and with a CTP analog. Nucleating ParB is an open clamp, in which parS is captured at the DNA-binding domain (the DNA-gate). Upon binding CTP, the N-terminal domain (NTD) self-dimerizes to close the NTD-gate of the clamp. The DNA-gate also closes, thus driving parS into a compartment between the DNA-gate and the C-terminal domain. CTP hydrolysis and/or the release of hydrolytic products are likely associated with reopening of the gates to release DNA and recycle ParB. Overall, we suggest a CTP-operated gating mechanism that regulates ParB nucleation, spreading, and recycling.
|
Aug 2021
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[18565, 25108]
Open Access
Abstract: ATP- and GTP-dependent molecular switches are extensively used to control functions of proteins in a wide range of biological processes. However, CTP switches are rarely reported. Here, we report that a nucleoid occlusion protein Noc is a CTPase enzyme whose membrane-binding activity is directly regulated by a CTP switch. In Bacillus subtilis, Noc nucleates on 16 bp NBS sites before associating with neighboring non-specific DNA to form large membrane-associated nucleoprotein complexes to physically occlude assembly of the cell division machinery. By in vitro reconstitution, we show that (1) CTP is required for Noc to form the NBS-dependent nucleoprotein complex, and (2) CTP binding, but not hydrolysis, switches Noc to a membrane-active state. Overall, we suggest that CTP couples membrane-binding activity of Noc to nucleoprotein complex formation to ensure productive recruitment of DNA to the bacterial cell membrane for nucleoid occlusion activity.
|
Jul 2021
|
|
I03-Macromolecular Crystallography
|
Aleksandra
Bialas
,
Thorsten
Langner
,
Adeline
Harant
,
Mauricio P.
Contreras
,
Clare E. M.
Stevenson
,
David M.
Lawson
,
Jan
Sklenar
,
Ronny
Kellner
,
Matthew J
Moscou
,
Ryohei
Terauchi
,
Mark J.
Banfield
,
Sophien
Kamoun
Diamond Proposal Number(s):
[18565]
Open Access
Abstract: A subset of plant NLR immune receptors carry unconventional integrated domains in addition to their canonical domain architecture. One example is rice Pik-1 that comprises an integrated heavy metal-associated (HMA) domain. Here, we reconstructed the evolutionary history of Pik-1 and its NLR partner, Pik-2, and tested hypotheses about adaptive evolution of the HMA domain. Phylogenetic analyses revealed that the HMA domain integrated into Pik-1 before Oryzinae speciation over 15 million years ago and has been under diversifying selection. Ancestral sequence reconstruction coupled with functional studies showed that two Pik-1 allelic variants independently evolved from a weakly binding ancestral state to high-affinity binding of the blast fungus effector AVR-PikD. We conclude that for most of its evolutionary history the Pik-1 HMA domain did not sense AVR-PikD, and that different Pik-1 receptors have recently evolved through distinct biochemical paths to produce similar phenotypic outcomes. These findings highlight the dynamic nature of the evolutionary mechanisms underpinning NLR adaptation to plant pathogens.
|
Jul 2021
|
|