I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[18548]
Open Access
Abstract: Interaction of BRCA2 through ca. 30 amino acid residue motifs, BRC repeats, with RAD51 is a conserved feature of the double-strand DNA break repair by homologous recombination in eukaryotes. In humans the binding of the eight BRC repeats is defined by two sequence motifs, FxxA and LFDE, interacting with distinct sites on RAD51. Little is known of the interaction of BRC repeats in other species, especially in protozoans, where variable number of BRC repeats are found in BRCA2 proteins. Here we have studied in detail the interactions of the two BRC repeats in Leishmania infantum BRCA2 with RAD51. We show LiBRC1 is a high-affinity repeat and determine the crystal structure of its complex with LiRAD51. Using truncation mutagenesis of the LiBRC1 repeat, we demonstrate that high affinity binding is maintained in the absence of an LFDE-like motif and suggest compensatory structural features. These observations point towards a divergent evolution of BRC repeats, where a common FxxA-binding ancestor evolved additional contacts for affinity maturation and fine-tuning.
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May 2022
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Amalyn
Nain-Perez
,
Anders
Foller Füchtbauer
,
Liliana
Håversen
,
Aleksei
Lulla
,
Chunxia
Gao
,
Josipa
Matic
,
Leticia
Monjas
,
Alexandra
Rodríguez
,
Paul
Brear
,
Woonghee
Kim
,
Marko
Hyvonen
,
Jan
Borén
,
Adil
Mardinoglu
,
Mathias
Uhlen
,
Morten
Grøtli
Diamond Proposal Number(s):
[25402]
Abstract: Liver pyruvate kinase (PKL) is a major regulator of metabolic flux and ATP production during liver cell glycolysis and is considered a potential drug target for the treatment of non-alcoholic fatty liver disease (NAFLD). In this study, we report the first ADP-competitive PKL inhibitors and identify several starting points for the further optimization of these inhibitors. Modeling and structural biology guided the optimization of a PKL-specific anthraquinone-based compound. A structure–activity relationship study of 47 novel synthetic derivatives revealed PKL inhibitors with half-maximal inhibitory concentration (IC50) values in the 200 nM range. Despite the difficulty involved in studying a binding site as exposed as the ADP site, these derivatives feature expanded structural diversity and chemical spaces that may be used to improve their inhibitory activities against PKL. The obtained results expand the knowledge of the structural requirements for interactions with the ADP-binding site of PKL.
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Mar 2022
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[25402]
Open Access
Abstract: The induction of interferon (IFN)-stimulated genes by STATs is a critical host defense mechanism against virus infection. Here, we report that a highly expressed poxvirus protein, 018, inhibits IFN-induced signaling by binding to the SH2 domain of STAT1, thereby preventing the association of STAT1 with an activated IFN receptor. Despite encoding other inhibitors of IFN-induced signaling, a poxvirus mutant lacking 018 was attenuated in mice. The 2.0 Å crystal structure of the 018:STAT1 complex reveals a phosphotyrosine-independent mode of 018 binding to the SH2 domain of STAT1. Moreover, the STAT1-binding motif of 018 shows similarity to the STAT1-binding proteins from Nipah virus, which, similar to 018, block the association of STAT1 with an IFN receptor. Overall, these results uncover a conserved mechanism of STAT1 antagonism that is employed independently by distinct virus families.
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Feb 2022
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18548]
Abstract: Exchanges of protein sequence modules support leaps in function unavailable through point mutations during evolution. Here we study the role of the two RAD51-interacting modules within the eight binding BRC repeats of BRCA2. We created 64 chimeric repeats by shuffling these modules and measured their binding to RAD51. We found that certain shuffled module combinations were stronger binders than any of the module combinations in the natural repeats. Surprisingly, the contribution from the two modules was poorly correlated with affinities of natural repeats, with a weak BRC8 repeat containing the most effective N-terminal module. The binding of the strongest chimera, BRC8-2, to RAD51 was improved by −2.4 kCal/mol compared to the strongest natural repeat, BRC4. A crystal structure of RAD51:BRC8-2 complex shows an improved interface fit and an extended β-hairpin in this repeat. BRC8-2 was shown to function in human cells, preventing the formation of nuclear RAD51 foci after ionizing radiation.
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Nov 2021
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Duncan E.
Scott
,
Nicola J.
Francis-Newton
,
May E.
Marsh
,
Anthony G.
Coyne
,
Gerhard
Fischer
,
Tommaso
Moschetti
,
Andrew R.
Bayly
,
Timothy D.
Sharpe
,
Kalina T.
Haas
,
Lorraine
Barber
,
Chiara R.
Valenzano
,
Rajavel
Srinivasan
,
David J.
Huggins
,
Miyoung
Lee
,
Amy
Emery
,
Bryn
Hardwick
,
Matthias
Ehebauer
,
Claudio
Dagostin
,
Alessandro
Esposito
,
Luca
Pellegrini
,
Trevor
Perrior
,
Grahame
Mckenzie
,
Tom L.
Blundell
,
Marko
Hyvonen
,
John
Skidmore
,
Ashok R.
Venkitaraman
,
Chris
Abell
Diamond Proposal Number(s):
[315, 7141]
Open Access
Abstract: BRCA2 controls RAD51 recombinase during homologous DNA recombination (HDR) through eight evolutionarily conserved BRC repeats, which individually engage RAD51 via the motif Phe-x-x-Ala. Using structure-guided molecular design, templated on a monomeric thermostable chimera between human RAD51 and archaeal RadA, we identify CAM833, a 529 Da orthosteric inhibitor of RAD51:BRC with a Kd of 366 nM. The quinoline of CAM833 occupies a hotspot, the Phe-binding pocket on RAD51 and the methyl of the substituted α-methylbenzyl group occupies the Ala-binding pocket. In cells, CAM833 diminishes formation of damage-induced RAD51 nuclear foci; inhibits RAD51 molecular clustering, suppressing extended RAD51 filament assembly; potentiates cytotoxicity by ionizing radiation, augmenting 4N cell-cycle arrest and apoptotic cell death and works with poly-ADP ribose polymerase (PARP)1 inhibitors to suppress growth in BRCA2-wildtype cells. Thus, chemical inhibition of the protein-protein interaction between BRCA2 and RAD51 disrupts HDR and potentiates DNA damage-induced cell death, with implications for cancer therapy.
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Mar 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[9537, 14043, 18548]
Abstract: CK2α is a ubiquitous, well-studied kinase that is a target for small-molecule inhibition, for treatment of cancers. While many different classes of adenosine 5′-triphosphate (ATP)-competitive inhibitors have been described for CK2α, they tend to suffer from significant off-target activity and new approaches are needed. A series of inhibitors of CK2α has recently been described as allosteric, acting at a previously unidentified binding site. Given the similarity of these inhibitors to known ATP-competitive inhibitors, we have investigated them further. In our thorough structural and biophysical analyses, we have found no evidence that these inhibitors bind to the proposed allosteric site. Rather, we report crystal structures, competitive isothermal titration calorimetry (ITC) and NMR, hydrogen–deuterium exchange (HDX) mass spectrometry, and chemoinformatic analyses that all point to these compounds binding in the ATP pocket. Comparisons of our results and experimental approach with the data presented in the original report suggest that the primary reason for the disparity is nonspecific inhibition by aggregation.
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Oct 2020
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Sarah L.
Kidd
,
Elaine
Fowler
,
Till
Reinhardt
,
Thomas
Compton
,
Natalia
Mateu
,
Hector
Newman
,
Dom
Bellini
,
Romain
Talon
,
Joseph
Mcloughlin
,
Tobias
Krojer
,
Anthony
Aimon
,
Anthony
Bradley
,
Michael
Fairhead
,
Paul
Brear
,
Laura
Diaz-Saez
,
Katherine
Mcauley
,
Hannah F.
Sore
,
Andrew
Madin
,
Daniel H.
O'Donovan
,
Kilian
Huber
,
Marko
Hyvonen
,
Frank
Von Delft
,
Christopher G.
Dowson
,
David R.
Spring
Diamond Proposal Number(s):
[18145, 15649, 14303, 14493]
Open Access
Abstract: Organic synthesis underpins the evolution of weak fragment hits into potent lead compounds. Deficiencies within current screening collections often result in the requirement of significant synthetic investment to enable multidirectional fragment growth, limiting the efficiency of the hit evolution process. Diversity-oriented synthesis (DOS)-derived fragment libraries are constructed in an efficient and modular fashion and thus are well-suited to address this challenge. To demonstrate the effective nature of such libraries within fragment-based drug discovery, we herein describe the screening of a 40-member DOS library against three functionally distinct biological targets using X-Ray crystallography. Firstly, we demonstrate the importance for diversity in aiding hit identification with four fragment binders resulting from these efforts. Moreover, we also exemplify the ability to readily access a library of analogues from cheap commercially available materials, which ultimately enabled the exploration of a minimum of four synthetic vectors from each molecule. In total, 10–14 analogues of each hit were rapidly accessed in three to six synthetic steps. Thus, we showcase how DOS-derived fragment libraries enable efficient hit derivatisation and can be utilised to remove the synthetic limitations encountered in early stage fragment-based drug discovery.
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May 2020
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I03-Macromolecular Crystallography
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Alexander V.
Strizhak
,
Oleg
Babii
,
Sergii
Afonin
,
Iuliia
Bakanovich
,
Teodors
Pantelejevs
,
Wenshu
Xu
,
Elaine
Fowler
,
Rohan
Eapen
,
Krishna
Sharma
,
Maxim O.
Platonov
,
Vasyl V.
Hurmach
,
Laura
Itzhaki
,
Marko
Hyvonen
,
Anne S.
Ulrich
,
David R.
Spring
,
Igor V.
Komarov
Diamond Proposal Number(s):
[18548]
Open Access
Abstract: Analogs of the known inhibitor (peptide pDI) of the p53/MDM2 protein–protein interaction are reported, which are stapled by linkers bearing a photoisomerizable diarylethene moiety. The corresponding photoisomers possess significantly different affinities to the p53-interacting domain of the human MDM2. Apparent dissociation constants are in the picomolar-to-low nanomolar range for those isomers with diarylethene in the “open” configuration, but up to eight times larger for the corresponding “closed” isomers. Spectroscopic, structural, and computational studies showed that the stapling linkers of the peptides contribute to their binding. Calorimetry revealed that the binding of the “closed” isomers is mostly enthalpy-driven, whereas the “open” photoforms bind to the protein stronger due to their increased binding entropy. The results suggest that conformational dynamics of the protein-peptide complexes may explain the differences in the thermodynamic profiles of the binding.
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May 2020
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Pooja
Sharma
,
Robert
Mahen
,
Maxim
Rossmann
,
Jamie E.
Stokes
,
Bryn
Hardwick
,
David J.
Huggins
,
Amy
Emery
,
Dominique L.
Kunciw
,
Marko
Hyvonen
,
David R.
Spring
,
Grahame J.
Mckenzie
,
Ashok R.
Venkitaraman
Diamond Proposal Number(s):
[7141, 9537]
Open Access
Abstract: The human polo-like kinase PLK1 coordinates mitotic chromosome segregation by phosphorylating multiple chromatin- and kinetochore-binding proteins. How PLK1 activity is directed to specific substrates via phosphopeptide recognition by its carboxyl-terminal polo-box domain (PBD) is poorly understood. Here, we combine molecular, structural and chemical biology to identify a determinant for PLK1 substrate recognition that is essential for proper chromosome segregation. We show that mutations ablating an evolutionarily conserved, Tyr-lined pocket in human PLK1 PBD trigger cellular anomalies in mitotic progression and timing. Tyr pocket mutations selectively impair PLK1 binding to the kinetochore phosphoprotein substrate PBIP1, but not to the centrosomal substrate NEDD1. Through a structure-guided approach, we develop a small-molecule inhibitor, Polotyrin, which occupies the Tyr pocket. Polotyrin recapitulates the mitotic defects caused by mutations in the Tyr pocket, further evidencing its essential function, and exemplifying a new approach for selective PLK1 inhibition. Thus, our findings support a model wherein substrate discrimination via the Tyr pocket in the human PLK1 PBD regulates mitotic chromosome segregation to preserve genome integrity.
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Nov 2019
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I04-Macromolecular Crystallography
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Krishna
Sharma
,
Alexander V.
Strizhak
,
Elaine
Fowler
,
Xuelu
Wang
,
Wenshu
Xu
,
Claus
Hatt Jensen
,
Yuteng
Wu
,
Hannah F.
Sore
,
Yu Heng
Lau
,
Marko
Hyvonen
,
Laura S.
Itzhaki
,
David R.
Spring
Diamond Proposal Number(s):
[14043]
Abstract: The Sondheimer dialkyne is extensively used in double strain-promoted azide–alkyne cycloadditions. This reagent suffers with poor water-solubility and rapidly decomposes in aqueous solutions. This intrinsically limits its application in biological systems, and no effective solutions are currently available. Herein, we report the development of novel highly water-soluble, stable, and azide-reactive strained dialkyne reagents. To demonstrate their extensive utility, we applied our novel dialkynes to a double strain-promoted macrocyclisation strategy to generate functionalised p53-based stapled peptides for inhibiting the oncogenic p53-MDM2 interaction. These functionalised stapled peptides bind MDM2 with low nanomolar affinity and show p53 activation in a cellular environment. Overall, our highly soluble, stable and azide-reactive dialkynes offer significant advantages over the currently used Sondheimer dialkyne, and could be utilised for numerous biological applications.
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Aug 2019
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