I03-Macromolecular Crystallography
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Nicholas G. S.
Mcgregor
,
Chi-Lin
Kuo
,
Thomas
Beenakker
,
Chun-Sing
Wong
,
Wendy A.
Offen
,
Zachary
Armstrong
,
Bobby I.
Florea
,
Jeroen D.
Codee
,
Herman S.
Overkleeft
,
Hans
Aerts
,
Gideon
Davies
Diamond Proposal Number(s):
[24948, 18598]
Open Access
Abstract: Exo-β-mannosidases are a broad class of stereochemically retaining hydrolases that are essential for the breakdown of complex carbohydrate substrates found in all kingdoms of life. Yet the detection of exo-β-mannosidases in complex biological samples remains challenging, necessitating the development of new methodologies. Cyclophellitol and its analogues selectively label the catalytic nucleophiles of retaining glycoside hydrolases, making them valuable tool compounds. Furthermore, cyclophellitol can be readily redesigned to enable the incorporation of a detection tag, generating activity-based probes (ABPs) that can be used to detect and identify specific glycosidases in complex biological samples. Towards the development of ABPs for exo-β-mannosidases, we present a concise synthesis of β-manno-configured cyclophellitol, cyclophellitol aziridine, and N-alkyl cyclophellitol aziridines. We show that these probes covalently label exo-β-mannosidases from GH families 2, 5, and 164. Structural studies of the resulting complexes support a canonical mechanism-based mode of action in which the active site nucleophile attacks the pseudo-anomeric centre to form a stable ester linkage, mimicking the glycosyl enzyme intermediate. Furthermore, we demonstrate activity- based protein profiling using an N-alkyl aziridine derivative by specifically labelling MANBA in mouse kidney tissue. Together, these results show that synthetic manno-configured cyclophellitol analogues hold promise for detecting exo-β-mannosidases in biological and biomedical research.
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Dec 2021
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14843, 18812]
Open Access
Abstract: Here, we combine the use of host screening, protein crystallography and QM/MM molecular dynamics simulations to investigate how the protein structure affects iminium catalysis by biotinylated secondary amines in a model 1,4 conjugate addition reaction. Monomeric streptavidin (M-Sav) lacks a quaternary structure and the solvent-exposed reaction site resulted in poor product conversion in the model reaction with low enantio- and regioselectivities. These parameters were much improved when the tetrameric host T-Sav was used; indeed, residues at the symmetrical subunit interface were proven to be critical for catalysis through a mutagenesis study. The use of QM/MM simulations and the asymmetric dimeric variant D-Sav revealed that both Lys121 residues which are located in the hosting and neighboring subunits play a critical role in controlling the stereoselectivity and reactivity. Lastly, the D-Sav template, though providing a lower conversion than that of the symmetric tetrameric counterpart, is likely a better starting point for future protein engineering because each surrounding residue within the asymmetric scaffold can be refined for secondary amine catalysis.
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Nov 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Maria
Schwarz
,
Rebecca F. M.
Eno
,
Stefanie
Freitag-Pohl
,
Christopher R.
Coxon
,
Hannah E.
Straker
,
David J.
Wortley
,
David J.
Hughes
,
Glynn
Mitchell
,
Jenny
Moore
,
Ian
Cummins
,
Nawaporn
Onkokesung
,
Melissa
Brazier-Hicks
,
Robert
Edwards
,
Ehmke
Pohl
,
Patrick G.
Steel
Diamond Proposal Number(s):
[24948]
Open Access
Abstract: The evolution and growth of multiple-herbicide resistance (MHR) in grass weeds continues to threaten global cereal production. While various processes can contribute to resistance, earlier work has identified the phi class glutathione-S-transferase (AmGSTF1) as a functional biomarker of MHR in black-grass (Alopecurus myosuroides). This study provides further insights into the role of AmGSTF1 in MHR using a combination of chemical and structural biology. Crystal structures of wild-type AmGSTF1, together with two specifically designed variants that allowed the co-crystal structure determination with glutathione and a glutathione adduct of the AmGSTF1 inhibitor 4-chloro-7-nitro-benzofurazan (NBD-Cl) were obtained. These studies demonstrated that the inhibitory activity of NBD-Cl was associated with the occlusion of the active site and the impediment of substrate binding. A search for other selective inhibitors of AmGSTF1, using ligand-fishing experiments, identified a number of flavonoids as potential ligands. Subsequent experiments using black-grass extracts discovered a specific flavonoid as a natural ligand of the recombinant enzyme. A series of related synthetic flavonoids was prepared and their binding to AmGSTF1 was investigated showing a high affinity for derivatives bearing a O-5-decyl-α-carboxylate. Molecular modelling based on high-resolution crystal structures allowed a binding pose to be defined which explained flavonoid binding specificity. Crucially, high binding affinity was linked to a reversal of the herbicide resistance phenotype in MHR black-grass. Collectively, these results present a nature-inspired new lead for the development of herbicide synergists to counteract MHR in weeds.
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Nov 2021
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[19844]
Open Access
Abstract: Developing stereoselective synthetic routes that are efficient and cost-effective allows easy access to biologically active molecules. Our previous syntheses of allele-selective bumped inhibitors of the Bromo and Extra-Terminal (BET) domain proteins, Brd2, Brd3, Brd4 and BrdT, required a wasteful, late-stage alkylation step and expensive chiral separation. To circumvent these limitations, we developed a route based on stereocontrolled alkylation of an N-Pf protected aspartic acid derivative that was used in a divergent, racemisation-free protocol to yield structurally diverse and enantiopure triazolodiazepines. With this approach, we synthesized bumped thienodiazepine-based BET inhibitor, ET-JQ1-OMe, in five steps and 99% ee without the need for chiral chromatography. Exquisite selectivity of ET-JQ1-OMe for Leu-Ala and Leu-Val mutants over wild-type bromodomain was established by isothermal titration calorimetry and X-ray crystallography. Our new approach provides unambiguous chemical evidence for the absolute stereochemistry of the active, allele-specific BET inhibitors and a viable route that will open wider access to this compound class.
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Aug 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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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[9948]
Open Access
Abstract: Enzyme transition-state mimics can act as powerful inhibitors and allow structural studies that report on the conformation of the transition-state. Here, mannoimidazole, a mimic of the transition state of mannosidase catalyzed hydrolysis of mannosides, is shown to bind in a B2,5 conformation on the Clostridium perfringens GH125 α-1,6-mannosidase, providing additional evidence of a OS2–B2,5–1S5 conformational itinerary for enzymes of this family.
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Aug 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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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[17118, 19754]
Abstract: We investigate the self-assembly of a palmitoylated (C16-chain at the N terminus) peptide fragment in comparison to the unlipidated peptide EELNRYY, a fragment of the gut hormone peptide PYY3–36. The lipopeptide C16-EELNRYY shows remarkable pH-dependent self-assembly above measured critical aggregation concentrations, forming fibrils at pH 7, but micelles at pH 10. The parent peptide does not show self-assembly behaviour. The lipopeptide forms hydrogels at sufficiently high concentration at pH 7, the dynamic mechanical properties of which were measured. We also show that the tyrosine functionality at the C terminus of EELNRYY can be used to enzymatically produce the pigment melanin. The enzyme tyrosinase oxidises tyrosine into 3,4-dihydroxyphenylalanine (DOPA), DOPA-quinone and further products, eventually forming eumelanin. This is a mechanism of photo-protection in the skin, for this reason controlling tyrosinase activity is a major target for skin care applications and EELNRYY has potential to be developed for such uses.
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Apr 2019
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[13587]
Open Access
Abstract: Xyloglucan (XyG) is a complex polysaccharide that is ubiquitous and often abundant in the cell walls of terrestrial plants. XyG metabolism is therefore a key component of the global carbon cycle, and hence XyG enzymology is of significant fundamental and applied importance in biomass conversion. To facilitate structure–function analyses of XyG-specific endo-glucanases, we have synthesized a 2′,4′-dinitrophenyl 2-deoxy-2-fluoro-β-glycoside mechanism-based inhibitor based on the highly branched XyG repeating motif XXXG (Xyl3Glc4: ([α-D-Xylp-(1→6)]-β-D-Glcp-(1→4)-[α-D-Xylp-(1→6)]-β-D-Glcp-(1→4)-[α-D-Xylp-(1→6)]-β-D-Glcp-(1→4)-D-Glcp. Key steps in the chemo-enzymatic synthesis included selective enzyme hydrolysis of XyG polysaccharide to produce the core heptasaccharide, per-O-acetylation, α-bromination, reductive glycal formation, electrophilic fluorination, SNAr glycosylation, and Zemplen deprotection. The resulting compound, XXXG(2F)-β-DNP, specifically labelled the active sites of several endo-(xylo)glucanases by accumulation of a covalent glycosyl-enzyme intermediate, as revealed by intact protein mass spectrometry. Crystallography of a complex with a Cellvibrio japonicus Glycoside Hydrolase Family 5 (GH5) endo-xyloglucanase corroborated the covalent nature of the intermediate, and further revealed the anticipated specificity for the catalytic nucleophile of this anomeric-configuration-retaining glycosidase. This specificity complements that of an analogous XXXG N-bromoacetylglycosylamine inhibitor, which labelled the catalytic acid–base sidechain in the same enzyme [Attia, et al., Biotechnol. Biofuels, 2018, 11, 45]. We anticipate that these inhibitors may find continued use in mechanistic analyses of endo-(xylo)glucanases from diverse GH families.
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Oct 2018
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Duncan C.
Miller
,
Mathew P.
Martin
,
Santosh
Adhikari
,
Alfie
Brennan
,
Jane A.
Endicott
,
Bernard T.
Golding
,
Ian R.
Hardcastle
,
Amy
Heptinstall
,
Stephen
Hobson
,
Claire
Jennings
,
Lauren
Molyneux
,
Yvonne
Ng
,
Stephen R.
Wedge
,
Martin
Noble
,
Celine
Cano
Open Access
Abstract: ATAD2 is an ATPase that is overexpressed in a variety of cancers and associated with a poor patient prognosis. This protein has been suggested to function as a cofactor for a range of transcription factors, including the proto-oncogene MYC and the androgen receptor. ATAD2 comprises an ATPase domain, implicated in chromatin remodelling, and a bromodomain which allows it to interact with acetylated histone tails. Dissection of the functional roles of these two domains would benefit from the availability of selective, cell-permeable pharmacological probes. An in silico evaluation of the 3D structures of various bromodomains suggested that developing small molecule ligands for the bromodomain of ATAD2 is likely to be challenging, although recent reports have shown that ATAD2 bromodomain ligands can be identified. We report a structure-guided fragment-based approach to identify lead compounds for ATAD2 bromodomain inhibitor development. Our findings indicate that the ATAD2 bromodomain can accommodate fragment hits (Mr < 200) that yield productive structure–activity relationships, and structure-guided design enabled the introduction of selectivity over BRD4.
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Feb 2018
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