I04-Macromolecular Crystallography
|
Nace
Zidar
,
Andrej
Emanuel Cotman
,
Wessel
Sinnige
,
Ondrej
Benek
,
Michaela
Barančokova
,
Anamarija
Zega
,
Lucija
Peterlin Mašič
,
Tihomir
Tomašič
,
Janez
Ilaš
,
Sara R.
Henderson
,
Julia E. A.
Mundy
,
Anthony
Maxwell
,
Clare E. M.
Stevenson
,
David M.
Lawson
,
Geert
Jan Sterk
,
Rodrigo
Tosso
,
Lucas
Gutierrez
,
Ricardo D.
Enriz
,
Danijel
Kikelj
Diamond Proposal Number(s):
[18565]
Open Access
Abstract: N-(Benzothiazole-2-yl)pyrrolamide DNA gyrase inhibitors with benzyl or phenethyl substituents attached to position 3 of the benzothiazole ring or to the carboxamide nitrogen atom were prepared and studied for their inhibition of Escherichia coli DNA gyrase by supercoiling assay. Compared to inhibitors bearing the substituents at position 4 of the benzothiazole ring, the inhibition was attenuated by moving the substituent to position 3 and further to the carboxamide nitrogen atom. A co-crystal structure of (Z)-3-benzyl-2-((4,5-dibromo-1H-pyrrole-2-carbonyl)imino)-2,3-dihydrobenzo[d]-thiazole-6-carboxylic acid (I) in complex with E. coli GyrB24 (ATPase subdomain) was solved, revealing the binding mode of this type of inhibitor to the ATP-binding pocket of the E. coli GyrB subunit. The key binding interactions were identified and their contribution to binding was rationalised by quantum theory of atoms in molecules (QTAIM) analysis. Our study shows that the benzyl or phenethyl substituents bound to the benzothiazole core interact with the lipophilic floor of the active site, which consists mainly of residues Gly101, Gly102, Lys103 and Ser108. Compounds with substituents at position 3 of the benzothiazole core were up to two orders of magnitude more effective than compounds with substituents at the carboxamide nitrogen. In addition, the 6-oxalylamino compounds were more potent inhibitors of E. coli DNA gyrase than the corresponding 6-acetamido analogues.
|
Jun 2024
|
|
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[25108]
Open Access
Abstract: In eukaryotes, targeted protein degradation (TPD) typically depends on a series of interactions among ubiquitin ligases that transfer ubiquitin molecules to substrates leading to degradation by the 26S proteasome. We previously identified that the bacterial effector protein SAP05 mediates ubiquitin-independent TPD. SAP05 forms a ternary complex via interactions with the von Willebrand Factor Type A (vWA) domain of the proteasomal ubiquitin receptor Rpn10 and the zinc-finger (ZnF) domains of the SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) and GATA BINDING FACTOR (GATA) transcription factors (TFs). This leads to direct TPD of the TFs by the 26S proteasome. Here, we report the crystal structures of the SAP05–Rpn10vWA complex at 2.17 Å resolution and of the SAP05–SPL5ZnF complex at 2.20 Å resolution. Structural analyses revealed that SAP05 displays a remarkable bimodular architecture with two distinct nonoverlapping surfaces, a “loop surface” with three protruding loops that form electrostatic interactions with ZnF, and a “sheet surface” featuring two β-sheets, loops, and α-helices that establish polar interactions with vWA. SAP05 binding to ZnF TFs involves single amino acids responsible for multiple contacts, while SAP05 binding to vWA is more stable due to the necessity of multiple mutations to break the interaction. In addition, positioning of the SAP05 complex on the 26S proteasome points to a mechanism of protein degradation. Collectively, our findings demonstrate how a small bacterial bimodular protein can bypass the canonical ubiquitin–proteasome proteolysis pathway, enabling ubiquitin-independent TPD in eukaryotic cells. This knowledge holds significant potential for the creation of TPD technologies.
|
Dec 2023
|
|
I04-Macromolecular Crystallography
|
Adam R.
Bentham
,
Juan Carlos
De La Concepcion
,
Javier Vega
Benjumea
,
Jiorgos
Kourelis
,
Sally
Jones
,
Melanie
Mendel
,
Jack
Stubbs
,
Clare E. M.
Stevenson
,
Josephine H. R.
Maidment
,
Mark
Youles
,
Rafal
Zdrzalek
,
Sophien
Kamoun
,
Mark J.
Banfield
Diamond Proposal Number(s):
[25108]
Open Access
Abstract: Engineering the plant immune system offers genetic solutions to mitigate crop diseases caused by diverse agriculturally significant pathogens and pests. Modification of intracellular plant immune receptors of the nucleotide-binding leucine rich repeat (NLR) superfamily for expanded recognition of pathogen virulence proteins (effectors) is a promising approach for engineering disease resistance. However, engineering can cause NLR autoactivation, resulting in constitutive defence responses that are deleterious to the plant. This may be due to plant NLRs associating in highly complex signalling networks that co-evolve together, and changes through breeding or genetic modification can generate incompatible combinations, resulting in autoimmune phenotypes. The sensor and helper NLRs of the rice (Oryza sativa) NLR pair Pik have co-evolved, and mismatching between non-co-evolved alleles triggers constitutive activation and cell death. This limits the extent to which protein modifications can be used to engineer pathogen recognition and enhance disease resistance mediated by these NLRs. Here, we dissected incompatibility determinants in the Pik pair in Nicotiana benthamiana and found that heavy metal-associated (HMA) domains integrated in Pik-1 not only evolved to bind pathogen effectors but also likely co-evolved with other NLR domains to maintain immune homeostasis. This explains why changes in integrated domains can lead to autoactivation. We then used this knowledge to facilitate engineering of new effector recognition specificities, overcoming initial autoimmune penalties. We show that by mismatching alleles of the rice sensor and helper NLRs Pik-1 and Pik-2, we can enable the integration of synthetic domains with novel and enhanced recognition specificities. Taken together, our results reveal a strategy for engineering NLRs, which has the potential to allow an expanded set of integrations and therefore new disease resistance specificities in plants.
|
Jul 2023
|
|
I03-Macromolecular Crystallography
|
Maša
Sterle
,
Martina
Durcik
,
Clare E. M.
Stevenson
,
Sara
Henderson
,
Petra Eva
Szili
,
Marton
Czikkely
,
David M.
Lawson
,
Anthony
Maxwell
,
Dominique
Cahard
,
Danijel
Kikelj
,
Nace
Zidar
,
Csaba
Pal
,
Lucija Peterlin
Mašič
,
Janez
Ilaš
,
Tihomir
Tomašič
,
Andrej Emanuel
Cotman
,
Anamarija
Zega
Diamond Proposal Number(s):
[18565]
Open Access
Abstract: We present a new series of 2-aminobenzothiazole-based DNA gyrase B inhibitors with promising activity against ESKAPE bacterial pathogens. Based on the binding information extracted from the cocrystal structure of DNA gyrase B inhibitor A, in complex with Escherichia coli GyrB24, we expanded the chemical space of the benzothiazole-based series to the C5 position of the benzothiazole ring. In particular, compound E showed low nanomolar inhibition of DNA gyrase (IC50 < 10 nM) and broad-spectrum antibacterial activity against pathogens belonging to the ESKAPE group, with the minimum inhibitory concentration < 0.03 μg/mL for most Gram-positive strains and 4–16 μg/mL against Gram-negative E. coli, Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae. To understand the binding mode of the synthesized inhibitors, a combination of docking calculations, molecular dynamics (MD) simulations, and MD-derived structure-based pharmacophore modeling was performed. The computational analysis has revealed that the substitution at position C5 can be used to modify the physicochemical properties and antibacterial spectrum and enhance the inhibitory potency of the compounds. Additionally, a discussion of challenges associated with the synthesis of 5-substituted 2-aminobenzothiazoles is presented.
|
Jun 2023
|
|
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]
Open Access
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
|
|