I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[25402]
Open Access
Abstract: For several centuries, microorganisms and enzymes have been used for many different applications. Although many enzymes with industrial applications have already been reported, different screening technologies, methods and approaches are constantly being developed in order to allow the identification of enzymes with even more interesting applications. In our work, we have performed data mining on the Chitinophaga sp. genome, a gram-negative bacterium isolated from a bacterial consortium of sugarcane bagasse isolated from an ethanol plant. The analysis of 8 Mb allowed the identification of the chtcp gene, previously annotated as putative Cht4039. The corresponding codified enzyme, denominated as ChtCP, showed the HEXXH conserved motif of family M32 from thermostable carboxypeptidases. After expression in E. coli, the recombinant enzyme was characterized biochemically. ChtCP showed the highest activity versus benziloxicarbonil Ala-Trp at pH 7.5, suggesting a preference for hydrophobic substrates. Surprisingly, the highest activity of ChtCP observed was between 55 °C and 75 °C, and 62% activity was still displayed at 100 °C. We observed that Ca2+, Ba2+, Mn2+ and Mg2+ ions had a positive effect on the activity of ChtCP, and an increase of 30 °C in the melting temperature was observed in the presence of Co2+. These features together with the structure of ChtCP at 1.2 Å highlight the relevance of ChtCP for further biotechnological applications.
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Feb 2021
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18548, 14043]
Abstract: Ral GTPases have been implicated as critical drivers of cell growth and metastasis in numerous Ras-driven cancers. We have previously reported stapled peptides, based on the Ral effector RLIP76, that can disrupt Ral signaling. Stapled peptides are short peptides that are locked into their bioactive form using a synthetic brace. Here, using an affinity maturation of the RLIP76 Ral-binding domain, we identified several sequence substitutions that together improve binding to Ral proteins by more than 20-fold. Hits from the selection were rigorously analyzed to determine the contributions of individual residues and two 1.5 Å co-crystal structures of the tightest-binding mutants in complex with RalB revealed key interactions. Insights gained from this maturation were used to design second-generation stapled peptides based on RLIP76 that exhibited vastly improved selectivity for Ral GTPases when compared to the first-generation lead peptide. The binding of second-generation peptides to Ral proteins was quantified and the binding site of the lead peptide on RalB was determined by NMR. Stapled peptides successfully competed with multiple Ral-effector interactions in cellular lysates. Our findings demonstrate how manipulation of a native binding partner can assist in the rational design of stapled peptide inhibitors targeting a protein–protein interaction.
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Nov 2020
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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
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[14043]
Abstract: The most abundant member of the collagen protein family, collagen I (also known as type I collagen; COL1), is composed of one unique (chain B) and two similar (chain A) polypeptides that self-assemble with one amino acid offset into a heterotrimeric triple helix. Given the offset, chain B can occupy either the leading (BAA), middle (ABA) or trailing (AAB) position of the triple helix, yielding three isomeric biomacromolecules with different protein recognition properties. Despite five decades of intensive research, there is no consensus on the position of chain B in COL1. Here, three triple-helical heterotrimers that each contain a putative von Willebrand factor (VWF) and discoidin domain receptor (DDR) recognition sequence from COL1 were designed with chain B permutated in all three positions. AAB demonstrated a strong preference for both VWF and DDR, and also induced higher levels of cellular DDR phosphorylation. Thus, we resolve this long-standing mystery and show that COL1 adopts an AAB register.
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Jan 2020
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Irina
Kufareva
,
Benoit
Bestgen
,
Paul
Brear
,
Renaud
Prudent
,
Béatrice
Laudet
,
Virginie
Moucadel
,
Mohamed
Ettaoussi
,
Celine F.
Sautel
,
Isabelle
Krimm
,
Matthias
Engel
,
Odile
Filhol
,
Marc
Le Borgne
,
Thierry
Lomberget
,
Claude
Cochet
,
Ruben
Abagyan
Diamond Proposal Number(s):
[18548]
Abstract: CK2 is a constitutively active protein kinase overexpressed in numerous malignancies. Interaction between CK2α and CK2β subunits is essential for substrate selectivity. The CK2α/CK2β interface has been previously targeted by peptides to achieve functional effects; however, no small molecules modulators were identified due to pocket flexibility and open shape. Here we generated numerous plausible conformations of the interface using the fumigation modeling protocol, and virtually screened a compound library to discover compound 1 that suppressed CK2α/CK2β interaction in vitro and inhibited CK2 in a substrate-selective manner. Orthogonal SPR, crystallography, and NMR experiments demonstrated that 4 and 6, improved analogs of 1, bind to CK2α as predicted. Both inhibitors alter CK2 activity in cells through inhibition of CK2 holoenzyme formation. Treatment with 6 suppressed MDA-MB231 triple negative breast cancer cell growth and induced apoptosis. Altogether, our findings exemplify an innovative computational-experimental approach and identify novel non-peptidic inhibitors of CK2 subunit interface disclosing substrate-selective functional effects.
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Nov 2019
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14043]
Abstract: Unlike many other well-characterized bacteria, the opportunistic human pathogen Pseudomonas aeruginosa relies exclusively on the Entner–Doudoroff pathway (EDP) for glycolysis. Pyruvate kinase (PK) is the main “pacemaker” of the EDP, and its activity is also relevant for P. aeruginosa virulence. Two distinct isozymes of bacterial PK have been recognized, PykA and PykF. Here, using growth and expression analyses of relevant PK mutants, we show that PykA is the dominant isoform in P. aeruginosa. Enzyme kinetics assays revealed that PykA displays potent K-type allosteric activation by glucose 6-phosphate and by intermediates from the pentose phosphate pathway. Unexpectedly, the X-ray structure of PykA at 2.4 Å resolution revealed that glucose 6-phosphate binds in a pocket that is distinct from the binding site reported for this metabolite in the PK from Mycobacterium tuberculosis (the only other available bacterial PK structure containing bound glucose 6-phosphate). We propose a mechanism by which glucose 6-phosphate binding at the allosteric site communicates with the PykA active site. Taken together, our findings indicate remarkable evolutionary plasticity in the mechanism(s) by which PK senses and responds to allosteric signals.
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Sep 2019
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18548]
Abstract: With the growing worldwide prevalence of antibiotic-resistant strains of tuberculosis (TB), new targets are urgently required for the development of treatments with novel modes of action. Fumarate hydratase (fumarase), a vulnerable component of the citric acid cycle in Mycobacterium tuberculosis (Mtb), is a metabolic target that could satisfy this unmet demand. A key challenge in the targeting of Mtb fumarase is its similarity to the human homolog, which shares an identical active site. A potential solution to this selectivity problem was previously found in a high-throughput screening hit that binds in a non-conserved allosteric site. In this work, a structure-activity relationship study was carried out with the determination of further structural biology on the lead series, affording derivatives with sub-micromolar inhibition. Further, the screening of this series against Mtb in vitro identified compounds with potent minimum inhibitory concentrations (MIC).
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Sep 2019
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18548]
Open Access
Abstract: The discovery of new Protein–Protein Interaction (PPI) modulators is currently limited by the difficulties associated with the design and synthesis of selective small molecule inhibitors. Peptides are a potential solution for disrupting PPIs; however, they typically suffer from poor stability in vivo and limited tissue penetration hampering their wide spread use as new chemical biology tools and potential therapeutics. In this work, a combination of CuAAC chemistry, molecular modelling, X-ray crystallography, and biological validation allowed us to develop highly functionalised peptide PPI inhibitors of the protein CK2. The lead peptide, CAM7117, prevents the formation of the holoenzyme assembly in vitro, slows down proliferation, induces apoptosis in cancer cells and is stable in human serum. CAM7117 could aid the development of novel CK2 inhibitors acting at the interface and help to fully understand the intracellular pathways involving CK2. Importantly, the approach adopted herein could be applied to many PPI targets and has the potential to ease the study of PPIs by efficiently providing access to functionalised peptides.
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Apr 2019
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[9537, 14043]
Abstract: The glyoxylate shunt bypasses the oxidative decarboxylation steps of the tricarboxylic acid (TCA) cycle, thereby conserving carbon skeletons for gluconeogenesis and biomass production. In Escherichia coli, carbon flux is redirected through the first enzyme of the glyoxylate shunt, isocitrate lyase (ICL), following phosphorylation and inactivation of the TCA cycle enzyme, isocitrate dehydrogenase (ICD), by the kinase/phosphatase, AceK. In contrast, mycobacterial species lack AceK and employ a phosphorylation-insensitive isocitrate dehydrogenase (IDH), which is allosterically activated by the product of ICL activity, glyoxylate. However, Pseudomonas aeruginosa, expresses IDH, ICD, ICL, and AceK, raising the question of how these enzymes are regulated to ensure proper flux distribution between the competing pathways. Here, we present the structure, kinetics, and regulation of ICL, IDH, and ICD from P. aeruginosa. We found that flux partitioning is coordinated through reciprocal regulation of these enzymes, linking distribution of carbon flux to availability of the key gluconeogenic precursors, oxaloacetate and pyruvate. Specifically, a greater abundance of these metabolites activated IDH and inhibited ICL, leading to increased TCA cycle flux. Regulation was also exerted through AceK-dependent phosphorylation of ICD; high levels of acetyl-CoA (which would be expected to accumulate when oxaloacetate is limiting) stimulated the kinase activity of AceK, whereas high levels of oxaloacetate stimulated its phosphatase activity. In summary, the TCA cycle–glyoxylate shunt branch-point in P. aeruginosa has a complex enzymology which is profoundly different to that in other species characterized to date. Presumably, this reflects its predilection for consuming fatty acids, especially during infection scenarios.
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Jul 2018
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