I04-1-Macromolecular Crystallography (fixed wavelength)
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Muhamamd
Faheem
,
Napoleao
Fonseca Valadares
,
Jose
Brandao-Neto
,
Domenico
Bellini
,
Patrick
Collins
,
Nicholas M.
Pearce
,
Louise
Bird
,
Juliana
Torini De Souza
,
Raymond
Owens
,
Humberto
Pereira
,
Frank
Von Delft
,
João Alexandre Ribeiro Gonçalves
Barbosa
Diamond Proposal Number(s):
[11175]
Open Access
Abstract: Several Schistosoma species cause Schistosomiasis, an endemic disease in 78 countries that is ranked second amongst the parasitic diseases in terms of its socioeconomic impact and human health importance. The drug recommended for treatment by the WHO is praziquantel (PZQ), but there are concerns associated with PZQ, such as the lack of information about its exact mechanism of action, its high price, its effectiveness – which is limited to the parasite’s adult form – and reports of resistance. The parasites lack the de novo purine pathway, rendering them dependent on the purine salvage pathway or host purine bases for nucleotide synthesis. Thus, the Schistosoma purine salvage pathway is an attractive target for the development of necessary and selective new drugs. In this study, the purine nucleotide phosphorylase II (PNP2), a new isoform of PNP1, was submitted to a high-throughput fragment-based hit discovery using a crystallographic screening strategy. PNP2 was crystallized and crystals were soaked with 827 fragments, a subset of the Maybridge 1000 library. X-ray diffraction data was collected and structures were solved. Out of 827-screened fragments we have obtained a total of 19 fragments that show binding to PNP2. 14 of these fragments bind to the active site of PNP2, while five were observed in three other sites. Here we present the first fragment screening against PNP2.
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Sep 2021
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I03-Macromolecular Crystallography
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Jessie
Branch
,
Badri S.
Rajagopal
,
Alessandro
Paradisi
,
Nick
Yates
,
Peter J
Lindley
,
Jake
Smith
,
Kristian
Hollingsworth
,
Bruce
Turnbull
,
Bernard
Henrissat
,
Alison
Parkin
,
Alan
Berry
,
Glyn R.
Hemsworth
Diamond Proposal Number(s):
[15378]
Open Access
Abstract: The release of glucose from lignocellulosic waste for subsequent fermentation into biofuels holds promise for securing humankind's future energy needs. The discovery of a set of copper dependent enzymes known as lytic polysaccharide monooxygenases (LPMOs) has galvanized new research in this area. LPMOs act by oxidatively introducing chain breaks into cellulose and other polysaccharides, boosting the ability of cellulases to act on the substrate. Although several proteins have been implicated as electron sources in fungal LPMO biochemistry, no equivalent bacterial LPMO electron donors have been previously identified, although the proteins Cbp2D and E from Cellvibrio japonicus have been implicated as potential candidates. Here we analyze a small c-type cytochrome (CjX183) present in Cellvibrio japonicus Cbp2D, and show that it caninitiate bacterial CuII/I LPMO reduction and also activate LPMO-catalyzed cellulose-degradation. In the absence of cellulose, CjX183-driven reduction of the LPMO results in less H2O2 production from O2, and correspondingly less oxidative damage to the enzyme than when ascorbate is used as the reducing agent. Significantly, using CjX183 as the activator maintained similar cellulase boosting levels relative to the use of an equivalent amount of ascorbate. Our results therefore add further evidence to the impact that the choice of electron source can have on LPMO action. Furthermore, the study of Cbp2D and other similar proteins may yet reveal new insight into the redox processes governing polysaccharide degradation in bacteria.
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Jul 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Open Access
Abstract: The human protein kinase ULK3 regulates the timing of membrane abscission, thus being involved in exosome budding and cytokinesis. Herein, we present the first high-resolution structures of the ULK3 kinase domain. Its unique features are explored against the background of other ULK kinases. An inhibitor fingerprint indicates that ULK3 is highly druggable and capable of adopting a wide range of conformations. In accordance with this, we describe a conformational switch between the active and an inactive ULK3 conformation, controlled by the properties of the attached small-molecule binder. Finally, we discuss a potential substrate-recognition mechanism of the full-length ULK3 protein.
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Jun 2021
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14631, 17180, 20015]
Open Access
Abstract: The activation loop (A-loop) plays a key role in regulating the catalytic activity of protein kinases. Phosphorylation in this region enhances the phosphoryl transfer rate of the kinase domain and increases its affinity for ATP. Furthermore, the A-loop possesses autoinhibitory functions in some kinases, where it collapses onto the protein surface and blocks substrate binding when unphosphorylated. Due to its flexible nature, the A-loop is usually disordered and untraceable in kinase domain crystal structures. The resulting lack of structural information is regrettable as it impedes the design of drug A-loop contacts, which have proven favourable in multiple cases. Here we characterize the binding with A-loop engagement between type 1.5 kinase inhibitor ‘example 172’ (EX172) and Mer tyrosine kinase (MerTK). With the help of crystal structures and binding kinetics we portray how the recruitment of the A-loop elicits a two-step binding mechanism which results in a drug-target complex characterized by high affinity and long residence time. In addition, the type 1.5 compound possesses excellent kinome selectivity and a remarkable preference for the phosphorylated over the dephosphorylated form of MerTK. We discuss these unique characteristics in the context of known type 1 and type 2 inhibitors and highlight opportunities for future kinase inhibitor design.
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Oct 2020
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Toxin-antitoxin systems play key roles in bacterial adaptation, including protection from antibiotic assault and infection by bacteriophages. The type IV toxin-antitoxin system AbiE encodes a DUF1814 nucleotidyltransferase-like toxin, and a two-domain antitoxin. In Streptococcus agalactiae, the antitoxin AbiEi negatively autoregulates abiE expression through positively co-operative binding to inverted repeats within the promoter. The human pathogen Mycobacterium tuberculosis encodes four DUF1814 putative toxins, two of which have antitoxins homologous to AbiEi. One such M.tuberculosis antitoxin, named Rv2827c, is required for growth and whilst the structure has previously been solved, the mode of regulation is unknown. To complete the gaps in our understanding, we first solved the structure of S. agalactiae AbiEi to 1.83 Å resolution for comparison with M.tuberculosis Rv2827c. AbiEi contains an N-terminal DNA binding domain and C-terminal antitoxicity domain, with bilateral faces of opposing charge. The overall AbiEi fold is similar to Rv2827c, though smaller, and with a 65º difference in C-terminal domain orientation. We further demonstrate that, like AbiEi, Rv2827c can autoregulate toxin-antitoxin operon expression. In contrast to AbiEi, the Prv2827c promoter contains two sets of inverted repeats, which bind Rv2827c with differing affinities depending on the sequence consensus. Surprisingly, Rv2827c bound with negative co-operativity to the full Prv2827c promoter, demonstrating an unexpectedly complex form of transcriptional regulation.
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Jun 2020
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[307, 10369, 19248]
Open Access
Abstract: Nek7 is a serine/threonine-protein kinase required for proper spindle formation and cytokinesis. Elevated Nek7 levels have been observed in several cancers, and inhibition of Nek7 might provide a route to the development of cancer therapeutics. To date, no selective and potent Nek7 inhibitors have been identified. Nek7 crystal structures exhibit an improperly formed regulatory-spine (R-spine), characteristic of an inactive kinase. We reasoned that the preference of Nek7 to crystallise in this inactive conformation might hinder attempts to capture Nek7 in complex with Type I inhibitors. Here, we have introduced aromatic residues into the R-spine of Nek7 with the aim to stabilise the active conformation of the kinase through R-spine stacking. The strong R-spine mutant Nek7SRS retained catalytic activity and was crystallised in complex with compound 51, an ATP-competitive inhibitor of Nek2 and Nek7. Subsequently, we obtained the same crystal form for wild-type Nek7WT in apo form and bound to compound 51. The R-spines of the three well-ordered Nek7WT molecules exhibit variable conformations while the R-spines of the Nek7SRS molecules all have the same, partially stacked configuration. Compound 51 bound to Nek2 and Nek7 in similar modes, but differences in the precise orientation of a substituent highlights features that could be exploited in designing inhibitors that are selective for particular Nek family members. Although the SRS mutations are not required to obtain a Nek7–inhibitor structure, we conclude that it is a useful strategy for restraining the conformation of a kinase in order to promote crystallogenesis.
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Apr 2020
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[17212]
Open Access
Abstract: Angiotensin-converting enzyme (ACE) is best known for its formation of the vasopressor angiotensin II that controls blood pressure but is also involved in other physiological functions through the hydrolysis of a variety of peptide substrates. The enzyme contains two catalytic domains (nACE and cACE) that have different affinities for ACE substrates and inhibitors. We investigated whether nACE inhibitor backbones contain a unique property which allows them to take advantage of the hinging of nACE. Kinetic analysis showed that mutation of unique nACE residues in both the S2 pocket and around the prime subsites (S′) to their C-domain counterparts each resulted in a decrease in affinity of nACE specific inhibitors (SG6, 33RE and ketoACE-13), but it required the combined S2_S′ mutant to abrogate nACE-selectivity. However, this was not observed with the non-domain-selective inhibitors enalaprilat and omapatrilat. High resolution structures were determined for the minimally glycosylated nACE with the combined S2_S′ mutations in complex with the ACE inhibitors 33RE (1.8 Å), omapatrilat (1.8 Å) and SG6 (1.7 Å). These confirmed that the affinities of the nACE-selective SG6, 33RE and ketoACE-13 are not only affected by direct interactions with the immediate environment of the binding site, but also by more distal residues. This study provides evidence for a more general mechanism of ACE inhibition involving synergistic effects of not only the S2, S1′ and S2′ subsites, but also residues involved in the subdomain interface that effect the unique ways in which the two domains stabilise active site loops to favour inhibitor binding.
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Mar 2020
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19880]
Open Access
Abstract: Attenuating the function of protein arginine methyltransferases (PRMTs) is an objective for the investigation and treatment of several diseases including cardiovascular disease and cancer. Bisubstrate inhibitors that simultaneously target binding sites for arginine substrate and the co-factor (S-adenosylmethionine (SAM)) have potential utility, but structural information on their binding is required for their development. Evaluation of bisubstrate inhibitors featuring an isosteric guanidine replacement with two prominent enzymes PRMT1 and CARM1 (PRMT4) by isothermal titration calorimetry (ITC), activity assays and crystallography are reported. Key findings are that 2-aminopyridine is a viable replacement for guanidine, providing an inhibitor that binds more strongly to CARM1 than PRMT1. Moreover, a residue around the active site that differs between CARM1 (Asn-265) and PRMT1 (Tyr-160) is identified that affects the side chain conformation of the catalytically important neighbouring glutamate in the crystal structures. Mutagenesis data supports its contribution to the difference in binding observed for this inhibitor. Structures of CARM1 in complex with a range of seven inhibitors reveal the binding modes and show that inhibitors with an amino acid terminus adopt a single conformation whereas the electron density for equivalent amine-bearing inhibitors is consistent with preferential binding in two conformations. These findings inform the molecular basis of CARM1 ligand binding and identify differences between CARM1 and PRMT1 that can inform drug discovery efforts.
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Feb 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Marcin
Bielecki
,
Svetlana
Antonyuk
,
Richard W.
Strange
,
Klaudia
Siemińska
,
John W.
Smalley
,
Paweł
Mackiewicz
,
Michał
Śmiga
,
Megan
Cowan
,
Michael J.
Capper
,
Paulina
Ślęzak
,
Mariusz
Olczak
,
Teresa
Olczak
Diamond Proposal Number(s):
[15991]
Abstract: As part of the infective process, Porphyromonas gingivalis must acquire heme which is indispensable for life and enables the microorganism to survive and multiply at the infection site. This oral pathogenic bacterium uses a newly discovered novel hmu heme uptake system with a leading role played by the HmuY hemophore-like protein, responsible for acquiring heme and increasing virulence of this periodontopathogen. We demonstrated that Prevotella intermedia produces two HmuY homologs, termed PinO and PinA. Both proteins were produced at higher mRNA and protein levels when the bacterium grew under low-iron/heme conditions. PinO and PinA bound heme, but preferentially under reducing conditions, and in a manner different to that of the P. gingivalis HmuY. The analysis of the three-dimensional structures confirmed differences between apo-PinO and apo-HmuY, mainly in the fold forming the heme-binding pocket. Instead of two histidine residues coordinating heme iron in P. gingivalis HmuY, PinO and PinA could use one methionine residue to fulfil this function, with potential support of additional methionine residue/s. The P. intermedia proteins sequestered heme only from the host albumin-heme complex under reducing conditions. Our findings suggest that HmuY-like family might comprise proteins subjected during evolution to significant diversification, resulting in different heme coordination mode. The newer data presented in this manuscript on HmuY homologs produced by P. intermedia sheds more light on the novel mechanism of heme uptake, could be helpful in discovering their biological function, and in developing novel therapeutic approaches.
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Jan 2020
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I03-Macromolecular Crystallography
I23-Long wavelength MX
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[12579]
Open Access
Abstract: Under limiting sulfur availability, bacteria can assimilate sulfur from alkanesulfonates. Bacteria utilize ATP-binding cassette (ABC) transporters to internalise them for further processing to release sulfur. In gram-negative bacteria the TauABC and SsuABC ensure internalization, although, these two systems have common substrates, the former has been characterized as a taurine specific system. TauA and SsuA are substrate-binding proteins (SBPs) that bind and bring the alkanesulfonates to the ABC importer for transport. Here, we have determined the crystal structure of TauA and have characterized its thermodynamic binding parameters by isothermal titration calorimetry in complex with taurine and different alkanesulfonates. Our structures revealed that the coordination of the alkanesulfonates is conserved, with the exception of Asp205 that is absent from SsuA, but the thermodynamic parameters revealed a very high enthalpic penalty cost for binding of the other alkanesulfonates relative to taurine. Our molecular dynamic simulations indicated that the different levels of hydration of the binding site contributed to the selectivity for taurine over the other alkanesulfonates. Such selectivity mechanism is very likely to be employed by other SBPs of ABC transporters.
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Nov 2019
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