I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[1225]
Open Access
Abstract: Trypanosomatids possess glycosome organelles that contain much of the glycolytic machinery, including phosphofructokinase (PFK). We present kinetic and structural data for PFK from three human pathogenic trypanosomatids, illustrating intriguing differences that may reflect evolutionary adaptations to differing ecological niches. The activity of Leishmania PFK – to a much larger extent than Trypanosoma PFK – is reliant on AMP for activity regulation, with 1 mm AMP increasing the L. infantum PFK (LiPFK) kcat/K0.5F6P value by 10‐fold, compared to only a 1.3‐ and 1.4‐fold increase for T. cruzi and T. brucei PFK, respectively. We also show that Leishmania PFK melts at a significantly lower (> 15 °C) temperature than Trypanosoma PFKs and that addition of either AMP or ATP results in a marked stabilization of the protein. Sequence comparisons of Trypanosoma spp. and Leishmania spp. show that divergence of the two genera involved amino acid substitutions that occur in the enzyme’s ‘reaching arms’ and ‘embracing arms’ that determine tetramer stability. The dramatic effects of AMP on Leishmania activity compared with the Trypanosoma PFKs may be explained by differences between the T‐to‐R equilibria for the two families, with the low‐melting Leishmania PFK favouring the flexible inactive T‐state in the absence of AMP. Sequence comparisons along with the enzymatic and structural data presented here also suggest there was a loss of AMP‐dependent regulation in Trypanosoma species rather than gain of this characteristic in Leishmania species and that AMP acts as a key regulator in Leishmania governing the balance between glycolysis and gluconeogenesis.
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Jan 2020
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I02-Macromolecular Crystallography
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Alice Rose
Mitchell
,
Meng
Yuan
,
Hugh P.
Morgan
,
Iain W.
Mcnae
,
Elizabeth A.
Blackburn
,
Thierry
Lebihan
,
R. A.
Homem
,
M.
Yu
,
Gary J.
Loake
,
Paul A.
Michels
,
Martin A.
Wear
,
Malcolm D.
Walkinshaw
Diamond Proposal Number(s):
[18515]
Open Access
Abstract: We show here that the M2 isoform of human pyruvate kinase (M2PYK) is susceptible to nitrosation and oxidation and that these modifications regulate enzyme activity by preventing formation of the active tetrameric form. The biotin switch assay carried out on M1 and M2 isoforms showed that M2PYK is sensitive to nitrosation and that Cys326 is highly susceptible to redox modification. Structural and enzymatic studies have been carried out on point mutants for three cysteine residues (Cys424, Cys358 and Cys326) to characterise their potential roles in redox regulation. Nine cysteines are conserved between M2PYK and M1PYK. Cys424 is the only cysteine unique to M2PYK. C424S, C424A, and C424L showed a moderate effect on enzyme activity with 80%, 100%, and 140% activity, respectively compared with M2PYK. C358 had been previously identified from in vivo studies to be the favoured target for oxidation. Our characterised mutant showed that this mutation stabilises tetrameric M2PYK suggesting that the in vivo resistance to oxidation for the Cys358Ser mutation is due to stabilisation of the tetrameric form of the enzyme. In contrast the Cys326Ser mutant exists predominantly in monomeric form. A biotin switch assay using this mutant also showed a significant reduction in biotinylation of M2PYK confirming that this is a major target for nitrosation and probably oxidation. Our results show that the sensitivity of M2PYK to oxidation and nitrosation is regulated by its monomer-tetramer equilibrium. In the monomer state, residues (in particular C326) are exposed to oxidative modifications that prevent reformation of the active tetrameric form.
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Sep 2018
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: We have tested the effect of all 20 proteinogenic amino acids on the activity of the M2 isoenzyme of pyruvate kinase (M2PYK) and show that within physiologically relevant concentrations, phenylalanine, alanine, tryptophan, methionine, valine, and proline act as inhibitors while histidine and serine act as activators. Size exclusion chromatography has been used to show that all amino acids, whether activators or inhibitors, stabilise the tetrameric form of M2PYK. In the absence of amino-acid ligands an apparent tetramer-monomer dissociation K d is estimated to be ~0.9 µM with a slow dissociation rate (t1/2 ~ 15 min). X-ray structures of M2PYK complexes with alanine, phenylalanine, and tryptophan show the M2PYK locked in an inactive T-state conformation, while activators lock the M2PYK tetramer in the active R-state conformation. Amino-acid binding in the allosteric pocket triggers rigid body rotations (11°) stabilising either T or R-states. The opposing inhibitory and activating effects of the non-essential amino acids serine and alanine suggest that M2PYK could act as a rapid-response nutrient sensor to rebalance cellular metabolism. This competition at a single allosteric site between activators and inhibitors provides a novel regulatory mechanism by which M2PYK activity is finely tuned by the relative (but not absolute) concentrations of activator and inhibitor amino acids. Such 'allostatic' regulation may be important in metabolic reprogramming and influencing cell fate.
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May 2018
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I04-Macromolecular Crystallography
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Pasquale
Linciano
,
Alice
Dawson
,
Ina
Pöhner
,
David M.
Costa
,
Monica S.
Sá
,
Anabela
Cordeiro-Da-Silva
,
Rosaria
Luciani
,
Sheraz
Gul
,
Gesa
Witt
,
Bernhard
Ellinger
,
Maria
Kuzikov
,
Philip
Gribbon
,
Jeanette
Reinshagen
,
Markus
Wolf
,
Birte
Behrens
,
Véronique
Hannaert
,
Paul A. M.
Michels
,
Erika
Nerini
,
Cecilia
Pozzi
,
Flavio
Di Pisa
,
Giacomo
Landi
,
Nuno
Santarem
,
Stefania
Ferrari
,
Puneet
Saxena
,
Sandra
Lazzari
,
Giuseppe
Cannazza
,
Lucio H.
Freitas-Junior
,
Carolina B.
Moraes
,
Bruno S.
Pascoalino
,
Laura M.
Alcântara
,
Claudia P.
Bertolacini
,
Vanessa
Fontana
,
Ulrike
Wittig
,
Wolfgang
Müller
,
Rebecca C.
Wade
,
William N.
Hunter
,
Stefano
Mangani
,
Luca
Costantino
,
Maria P.
Costi
Diamond Proposal Number(s):
[8574]
Open Access
Abstract: Pteridine reductase-1 (PTR1) is a promising drug target for the treatment of trypanosomiasis. We investigated the potential of a previously identified class of thiadiazole inhibitors of Leishmania major PTR1 for activity against Trypanosoma brucei (Tb). We solved crystal structures of several TbPTR1-inhibitor complexes to guide the structure-based design of new thiadiazole derivatives. Subsequent synthesis and enzyme- and cell-based assays confirm new, mid-micromolar inhibitors of TbPTR1 with low toxicity. In particular, compound 4m, a biphenyl-thiadiazole-2,5-diamine with IC50 = 16 μM, was able to potentiate the antitrypanosomal activity of the dihydrofolate reductase inhibitor methotrexate (MTX) with a 4.1-fold decrease of the EC50 value. In addition, the antiparasitic activity of the combination of 4m and MTX was reversed by addition of folic acid. By adopting an efficient hit discovery platform, we demonstrate, using the 2-amino-1,3,4-thiadiazole scaffold, how a promising tool for the development of anti-T. brucei agents can be obtained.
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Sep 2017
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13550]
Open Access
Abstract: The gluconeogenic enzyme fructose-1,6-bisphosphatase has been proposed as a potential drug target against Leishmania parasites that cause up to 20,000–30,000 deaths annually. A comparison of three crystal structures of L. major fructose-1,6-bisphosphatase (LmFBPase) along with enzyme kinetic data show how AMP acts as an allosteric inhibitor and provides insight into its metal-dependent reaction mechanism. The crystal structure of the apoenzyme form of LmFBPase is a homotetramer in which the dimer of dimers adopts a planar conformation with disordered ‘dynamic loops’. The structure of LmFBPase, complexed with manganese and its catalytic product phosphate shows the dynamic loops locked into the active sites. A third crystal structure of LmFBPase complexed with its allosteric inhibitor AMP shows an inactive form of the tetramer, in which the dimer pairs are rotated by 18° relative to each other. The three structures suggest an allosteric mechanism in which AMP binding triggers a rearrangement of hydrogen-bonds across the large and small interfaces. Retraction of the ‘effector loop’ required for AMP binding releases the side chain of His23 from the dimer-dimer interface. This is coupled with a flip of the side chain of Arg48 which ties down the key catalytic dynamic loop in a disengaged conformation and also locks the tetramer in an inactive rotated T-state. The structure of the effector site of LmFBPase shows different structural features compared with human FBPases, thereby offering a potential and species-specific drug target.
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Sep 2017
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Abstract: Bioinformatics studies have shown that the genomes of trypanosomatid species each encode one SCP2-thiolase-like protein (SLP), which is characterized by having the YDCF thiolase sequence fingerprint of the Cβ2-Cα2 loop. SLPs are only encoded by the genomes of these parasitic protists and not by those of mammals, including human. Deletion of the Trypanosoma brucei SLP gene (TbSLP) increases the doubling time of procyclic T. brucei and causes a 5-fold reduction of de novo sterol biosynthesis from glucose- and acetate-derived acetyl-CoA. Fluorescence analyses of EGFP-tagged TbSLP expressed in the parasite located the TbSLP in the mitochondrion. The crystal structure of TbSLP (refined at 1.75 Å resolution) confirms that TbSLP has the canonical dimeric thiolase fold. In addition, the structures of the TbSLP-acetoacetyl-CoA (1.90 Å) and TbSLP-malonyl-CoA (2.30 Å) complexes reveal that the two oxyanion holes of the thiolase active site are preserved. TbSLP binds malonyl-CoA tightly (Kd 90 µM), acetoacetyl-CoA moderately (Kd 0.9 mM) and acetyl-CoA and CoA very weakly. TbSLP possesses low malonyl-CoA decarboxylase activity. Altogether, the data show that TbSLP is a mitochondrial enzyme involved in lipid metabolism.
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Aug 2016
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[7613]
Open Access
Abstract: The transition between the inactive T-state (apoenzyme) and active R-state (effector bound enzyme) of Trypanosoma cruzi pyruvate kinase (PYK) is accompanied by a symmetrical 8° rigid body rocking motion of the A- and C-domain cores in each of the four subunits, coupled with the formation of additional salt bridges across two of the four subunit interfaces. These salt bridges provide increased tetramer stability correlated with an enhanced specificity constant (kcat/S0.5). A detailed kinetic and structural comparison between the potential drug target PYKs from the pathogenic protists T. cruzi, T. brucei and Leishmania mexicana shows that their allosteric mechanism is conserved. By contrast, a structural comparison of trypanosomatid PYKs with the evolutionarily divergent PYKs of humans and of bacteria shows that they have adopted different allosteric strategies. The underlying principle in each case is to maximize (kcat/S0.5) by stabilizing and rigidifying the tetramer in an active R-state conformation. However, bacterial and mammalian PYKs have evolved alternative ways of locking the tetramers together. In contrast to the divergent allosteric mechanisms, the PYK active sites are highly conserved across species. Selective disruption of the varied allosteric mechanisms may therefore provide a useful approach for the design of species-specific inhibitors.
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Sep 2014
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[7613]
Abstract: The phosphotransfer mechanism of PYKs (pyruvate kinases) has been studied in detail, but the mechanism of the intrinsic decarboxylase reaction catalysed by PYKs is still unknown. 1H NMR was used in the present study to follow OAA (oxaloacetate) decarboxylation by trypanosomatid and human PYKs confirming that the decarboxylase activity is conserved across distantly related species. Crystal structures of TbPYK (Trypanosoma brucei PYK) complexed with the product of the decarboxylase reaction (pyruvate), and a series of substrate analogues (D-malate, 2-oxoglutarate and oxalate) show that the OAA analogues bind to the kinase active site with similar binding modes, confirming that both decarboxylase and kinase activities share a common site for substrate binding and catalysis. Decarboxylation of OAA as monitored by NMR for TbPYK has a relatively low turnover with values of 0.86 s−1 and 1.47 s−1 in the absence and presence of F26BP (fructose 2,6-bisphosphate) respectively. Human M1PYK (M1 isoform of PYK) has a measured turnover value of 0.50 s−1. The X-ray structures explain why the decarboxylation activity is specific for OAA and is not general for α-oxo acid analogues. Conservation of the decarboxylase reaction across divergent species is a consequence of piggybacking on the conserved kinase mechanism which requires a stabilized enol intermediate.
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Mar 2014
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Abstract: The active site of pyruvate kinase (PYK) is located between the AC core of the enzyme and a mobile lid corresponding to domain B. Many PYK structures have already been determined, but the first `effector-only' structure and the first with PEP (the true natural substrate) are now reported for the enzyme from Trypanosoma brucei. PEP soaked into crystals of the enzyme with bound allosteric activator fructose 2,6-bisphosphate (F26BP) and Mg2+ triggers a substantial 23° rotation of the B domain `in crystallo', resulting in a partially closed active site. The interplay of side chains with Mg2+ and PEP may explain the mechanism of the domain movement. Furthermore, it is apparent that when F26BP is present but PEP is absent Mg2+ occupies a position that is distinct from the two canonical Mg2+-binding sites at the active site. This third site is adjacent to the active site and involves the same amino-acid side chains as in canonical site 1 but in altered orientations. Site 3 acts to sequester Mg2+ in a `priming' position such that the enzyme is maintained in its R-state conformation. In this way, Mg2+ cooperates with F26BP to ensure that the enzyme is in a conformation that has a high affinity for the substrate.
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May 2013
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I03-Macromolecular Crystallography
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Hugh
Morgan
,
Martin J.
Walsh
,
Elizabeth A.
Blackburn
,
Martin A.
Wear
,
Matthew B.
Boxer
,
Min
Shen
,
Iain W.
Mcnae
,
Matthew W.
Nowicki
,
Paul A. M.
Michels
,
Douglas
Auld
,
Linda
Fothergill-Gilmore
,
Malcolm D.
Walkinshaw
,
Henrike
Veith
Diamond Proposal Number(s):
[7613]
Abstract: PYK (pyruvate kinase) plays a central role in the metabolism of many organisms and cell types, but the elucidation of the details of its function in a systems biology context has been hampered by the lack of specific high-affinity small-molecule inhibitors. High-throughput screening has been used to identify a family of saccharin derivatives which inhibit LmPYK (Leishmania mexicana PYK) activity in a time- (and dose-) dependent manner, a characteristic of irreversible inhibition. The crystal structure of DBS {4-[(1,1-dioxo-1,2-benzothiazol-3-yl)sulfanyl]benzoic acid} complexed with LmPYK shows that the saccharin moiety reacts with an active-site lysine residue (Lys335), forming a covalent bond and sterically hindering the binding of ADP/ATP. Mutation of the lysine residue to an arginine residue eliminated the effect of the inhibitor molecule, providing confirmation of the proposed inhibitor mechanism. This lysine residue is conserved in the active sites of the four human PYK isoenzymes, which were also found to be irreversibly inhibited by DBS. X-ray structures of PYK isoforms show structural differences at the DBS-binding pocket, and this covalent inhibitor of PYK provides a chemical scaffold for the design of new families of potentially isoform-specific irreversible inhibitors.
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Sep 2012
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