I03-Macromolecular Crystallography
|
Open Access
Abstract: Transient Receptor Potential (TRP) channels have important roles in environmental sensing in animals. Human TRP subfamily A member 1 (TRPA1) is responsible for sensing allyl isothiocyanate (AITC) and other electrophilic sensory irritants. TRP subfamily vanilloid member 3 (TRPV3) is involved in skin maintenance. TRPV3 is a reported substrate of the 2-oxoglutarate oxygenase factor inhibiting hypoxia inducible factor (FIH). We report biochemical and structural studies concerning asparaginyl hydroxylation of the ankyrin repeat domains (ARDs) of TRPA1 and TRPV3 catalysed by FIH. The results with ARD peptides support a previous report on FIH-catalysed TRPV3 hydroxylation and show that, of the 12 potential TRPA1 sequences investigated, one sequence (TRPA1 residues 322-348) undergoes hydroxylation at Asn-336. Structural studies reveal that the TRPA1 and TRPV3 ARDs bind to FIH with a similar overall geometry to most other reported FIH substrates. However, the binding mode of TRPV3 to FIH is distinct from that of other substrates.
|
Nov 2022
|
|
I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Raphael
Reinbold
,
Ingvild C.
Hvinden
,
Patrick
Rabe
,
Ryan A.
Herold
,
Alina
Finch
,
James
Wood
,
Melissa
Morgan
,
Maximillian
Staudt
,
Ian J.
Clifton
,
Fraser A.
Armstrong
,
James S. O.
Mccullagh
,
Jo
Redmond
,
Chiara
Bardella
,
Martine I.
Abboud
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[23459]
Open Access
Abstract: Ivosidenib, an inhibitor of isocitrate dehydrogenase 1 (IDH1) R132C and R132H variants, is approved for the treatment of acute myeloid leukaemia (AML). Resistance to ivosidenib due to a second site mutation of IDH1 R132C, leading to IDH1 R132C/S280F, has emerged. We describe biochemical, crystallographic, and cellular studies on the IDH1 R132C/S280F and R132H/S280F variants that inform on the mechanism of second-site resistance, which involves both modulation of inhibitor binding at the IDH1 dimer-interface and alteration of kinetic properties, which enable more efficient 2-HG production relative to IDH1 R132C and IDH1 R132H. Importantly, the biochemical and cellular results demonstrate that it should be possible to overcome S280F mediated resistance in AML patients by using alternative inhibitors, including some presently in phase 2 clinical trials.
|
Aug 2022
|
|
I03-Macromolecular Crystallography
|
Patrick
Rabe
,
Carla C.
Walla
,
Noelle K.
Goodyear
,
Jordan
Welsh
,
Rebecca
Southwart
,
Ian
Clifton
,
James D. S.
Linyard
,
Anthony
Tumber
,
Tim D. W.
Claridge
,
William K.
Myers
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[23459]
Open Access
Abstract: Isopenicillin N synthase (IPNS) catalyses formation of the β-lactam and thiazolidine rings of isopenicillin N (IPN) from its linear tripeptide L-δ-(α-aminoadipoyl)-L-cysteinyl-D-valine (ACV) substrate in an iron and dioxygen (O2) dependent four electron oxidation without precedent in current synthetic chemistry. Recent X-ray free electron laser (XFEL) studies including time-resolved serial femtosecond crystallography show binding of O2 to the IPNS:Fe(II):ACV complex induces unexpected conformational changes in α-helices on the surface of IPNS, in particular in α3 and α10. However, how substrate binding leads to conformational changes away from the active site is unknown. Here, using detailed 19F NMR and EPR experiments with labelled IPNS variants, we investigated motions in α3 and α10 induced by binding of ferrous iron, ACV and the O2 analogue NO, using the less mobile α6 for comparison. 19F NMR studies were carried out on singly and doubly labelled α3, α6 and α10 variants at different temperatures. In addition, double electron-electron resonance (DEER) EPR analysis was carried out on doubly spin labelled variants. The combined spectroscopic and crystallographic results reveal that substantial conformational changes in regions of IPNS including α3 and α10 are induced by binding of ACV and NO. Since IPNS is a member of the structural superfamily of 2-oxoglutarate dependent oxygenases and related enzymes, related conformational changes may be of general importance in non-heme oxygenase catalysis.
|
Jul 2022
|
|
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Patrick
Rabe
,
Jos J. A. G.
Kamps
,
Kyle D.
Sutherlin
,
James D. S.
Linyard
,
Pierre
Aller
,
Cindy C.
Pham
,
Mikako
Makita
,
Ian
Clifton
,
Michael A.
Mcdonough
,
Thomas M.
Leissing
,
Denis
Shutin
,
Pauline A.
Lang
,
Agata
Butryn
,
Jurgen
Brem
,
Sheraz
Gul
,
Franklin D.
Fuller
,
In-Sik
Kim
,
Mun Hon
Cheah
,
Thomas
Fransson
,
Asmit
Bhowmick
,
Iris D.
Young
,
Lee
O'Riordan
,
Aaron S.
Brewster
,
Ilaria
Pettinati
,
Margaret
Doyle
,
Yasumasa
Joti
,
Shigeki
Owada
,
Kensuke
Tono
,
Alexander
Batyuk
,
Mark S.
Hunter
,
Roberto
Alonso-Mori
,
Uwe
Bergmann
,
Robin L.
Owen
,
Nicholas K.
Sauter
,
Timothy D. W.
Claridge
,
Carol V.
Robinson
,
Vittal K.
Yachandra
,
Junko
Yano
,
Jan F.
Kern
,
Allen M.
Orville
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[23459, 19458]
Open Access
Abstract: Isopenicillin N synthase (IPNS) catalyzes the unique reaction of L-δ-(α-aminoadipoyl)-L-cysteinyl-D-valine (ACV) with dioxygen giving isopenicillin N (IPN), the precursor of all natural penicillins and cephalosporins. X-ray free-electron laser studies including time-resolved crystallography and emission spectroscopy reveal how reaction of IPNS:Fe(II):ACV with dioxygen to yield an Fe(III) superoxide causes differences in active site volume and unexpected conformational changes that propagate to structurally remote regions. Combined with solution studies, the results reveal the importance of protein dynamics in regulating intermediate conformations during conversion of ACV to IPN. The results have implications for catalysis by multiple IPNS-related oxygenases, including those involved in the human hypoxic response, and highlight the power of serial femtosecond crystallography to provide insight into long-range enzyme dynamics during reactions presently impossible for nonprotein catalysts.
|
Aug 2021
|
|
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Tzu-Lan
Yeh
,
Thomas m.
Leissing
,
Martine I.
Abboud
,
Cyrille C.
Thinnes
,
Onur
Atasoylu
,
James P.
Holt-Martyn
,
Dong
Zhang
,
Anthony
Tumber
,
Kerstin
Lippl
,
Christopher T.
Lohans
,
Ivanhoe K. H.
Leung
,
Helen
Morcrette
,
Ian J.
Clifton
,
Timothy D. W.
Claridge
,
Akane
Kawamura
,
Emily
Flashman
,
Xin
Lu
,
Peter J.
Ratcliffe
,
Rasheduzzaman
Chowdhury
,
Christopher W.
Pugh
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[12346, 9306]
Open Access
Abstract: Inhibition of the human 2-oxoglutarate (2OG) dependent hypoxia inducible factor (HIF) prolyl hydroxylases (human PHD1–3) causes upregulation of HIF, thus promoting erythropoiesis and is therefore of therapeutic interest. We describe cellular, biophysical, and biochemical studies comparing four PHD inhibitors currently in clinical trials for anaemia treatment, that describe their mechanisms of action, potency against isolated enzymes and in cells, and selectivities versus representatives of other human 2OG oxygenase subfamilies. The ‘clinical’ PHD inhibitors are potent inhibitors of PHD catalyzed hydroxylation of the HIF-α oxygen dependent degradation domains (ODDs), and selective against most, but not all, representatives of other human 2OG dependent dioxygenase subfamilies. Crystallographic and NMR studies provide insights into the different active site binding modes of the inhibitors. Cell-based results reveal the inhibitors have similar effects on the upregulation of HIF target genes, but differ in the kinetics of their effects and in extent of inhibition of hydroxylation of the N- and C-terminal ODDs; the latter differences correlate with the biophysical observations.
|
Sep 2017
|
|
I04-Macromolecular Crystallography
|
Christopher T.
Lohans
,
David Y.
Wang
,
Christian
Jorgensen
,
Samuel T.
Cahill
,
Ian J.
Clifton
,
Michael A.
Mcdonough
,
Henry P.
Oswin
,
James
Spencer
,
Carmen
Domene
,
Timothy D. W.
Claridge
,
Jurgen
Brem
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[12346]
Open Access
Abstract: The class D (OXA) serine β-lactamases are a major cause of resistance to β-lactam antibiotics. The class D enzymes are unique amongst β-lactamases because they have a carbamylated lysine that acts as a general acid/base in catalysis. Previous crystallographic studies led to the proposal that β-lactamase inhibitor avibactam targets OXA enzymes in part by promoting decarbamylation. Similarly, halide ions are proposed to inhibit OXA enzymes via decarbamylation. NMR analyses, in which the carbamylated lysines of OXA-10, -23 and -48 were 13C-labelled, indicate that reaction with avibactam does not ablate lysine carbamylation in solution. While halide ions did not decarbamylate the 13C-labelled OXA enzymes in the absence of substrate or inhibitor, avibactam-treated OXA enzymes were susceptible to decarbamylation mediated by halide ions, suggesting halide ions may inhibit OXA enzymes by promoting decarbamylation of acyl-enzyme complex. Crystal structures of the OXA-10 avibactam complex were obtained with bromide, iodide, and sodium ions bound between Trp-154 and Lys-70. Structures were also obtained wherein bromide and iodide ions occupy the position expected for the ‘hydrolytic water’ molecule. In contrast with some solution studies, Lys-70 was decarbamylated in these structures. These results reveal clear differences between crystallographic and solution studies on the interaction of class D β-lactamases with avibactam and halides, and demonstrate the utility of 13C-NMR for studying lysine carbamylation in solution.
|
Jun 2017
|
|
I04-Macromolecular Crystallography
|
Guo-Bo
Li
,
Jurgen
Brem
,
Robert
Lesniak
,
Martine I.
Abboud
,
Christopher T.
Lohans
,
Ian J.
Clifton
,
Sheng-Yong
Yang
,
Juan-Carlos
Jiménez-Castellanos
,
Matthew B.
Avison
,
James
Spencer
,
Michael A.
Mcdonough
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[12346]
Abstract: Crystallographic analyses of the VIM-5 metallo-β-lactamase (MBL) with isoquinoline inhibitors reveal non zinc ion binding modes. Comparison with other MBL–inhibitor structures directed addition of a zinc-binding thiol enabling identification of potent B1 MBL inhibitors. The inhibitors potentiate meropenem activity against clinical isolates harboring MBLs.
|
Apr 2017
|
|
I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[12346]
Open Access
Abstract: β-Lactamases enable resistance to almost all β-lactam antibiotics. Pioneering work revealed that acyclic boronic acids can act as ‘transition state analogue’ inhibitors of nucleophilic serine enzymes, including serine-β-lactamases. Here we report biochemical and biophysical analyses revealing that cyclic boronates potently inhibit both nucleophilic serine and zinc-dependent β-lactamases by a mechanism involving mimicking of the common tetrahedral intermediate. Cyclic boronates also potently inhibit the non-essential penicillin-binding protein PBP 5 by the same mechanism of action. The results open the way for development of dual action inhibitors effective against both serine- and metallo-β-lactamases, and which could also have antimicrobial activity through inhibition of PBPs.
|
Aug 2016
|
|
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
|
Rasheduzzaman
Chowdhury
,
Rok
Sekirnik
,
Nigel C.
Brissett
,
Tobias
Krojer
,
Chia-Hua
Ho
,
Stanley S.
Ng
,
Ian J.
Clifton
,
Wei
Ge
,
Nadia J.
Kershaw
,
Gavin C.
Fox
,
Joao
Muniz
,
Melanie
Vollmar
,
Claire
Phillips
,
Ewa S.
Pilka
,
Kathryn L.
Kavanagh
,
Frank
Von Delft
,
Udo
Oppermann
,
Michael
Mcdonough
,
Aidan J.
Doherty
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[7495]
Abstract: 2-Oxoglutarate (2OG)-dependent oxygenases have important roles in the regulation of gene expression via demethylation of N-methylated chromatin components1, 2 and in the hydroxylation of transcription factors3 and splicing factor proteins4. Recently, 2OG-dependent oxygenases that catalyse hydroxylation of transfer RNA5, 6, 7 and ribosomal proteins8 have been shown to be important in translation relating to cellular growth, TH17-cell differentiation and translational accuracy9, 10, 11, 12. The finding that ribosomal oxygenases (ROXs) occur in organisms ranging from prokaryotes to humans8 raises questions as to their structural and evolutionary relationships. In Escherichia coli, YcfD catalyses arginine hydroxylation in the ribosomal protein L16; in humans, MYC-induced nuclear antigen (MINA53; also known as MINA) and nucleolar protein 66 (NO66) catalyse histidine hydroxylation in the ribosomal proteins RPL27A and RPL8, respectively. The functional assignments of ROXs open therapeutic possibilities via either ROX inhibition or targeting of differentially modified ribosomes. Despite differences in the residue and protein selectivities of prokaryotic and eukaryotic ROXs, comparison of the crystal structures of E. coli YcfD and Rhodothermus marinus YcfD with those of human MINA53 and NO66 reveals highly conserved folds and novel dimerization modes defining a new structural subfamily of 2OG-dependent oxygenases. ROX structures with and without their substrates support their functional assignments as hydroxylases but not demethylases, and reveal how the subfamily has evolved to catalyse the hydroxylation of different residue side chains of ribosomal proteins. Comparison of ROX crystal structures with those of other JmjC-domain-containing hydroxylases, including the hypoxia-inducible factor asparaginyl hydroxylase FIH and histone Nε-methyl lysine demethylases, identifies branch points in 2OG-dependent oxygenase evolution and distinguishes between JmjC-containing hydroxylases and demethylases catalysing modifications of translational and transcriptional machinery. The structures reveal that new protein hydroxylation activities can evolve by changing the coordination position from which the iron-bound substrate-oxidizing species reacts. This coordination flexibility has probably contributed to the evolution of the wide range of reactions catalysed by oxygenases.
|
May 2014
|
|
I04-Macromolecular Crystallography
|
Abstract: The hypoxic response in animals is mediated via the transcription factor hypoxia-inducible factor (HIF). An oxygen-sensing component of the HIF system is provided by Fe(II) and 2-oxoglutarate-dependent oxygenases that catalyse the posttranslational hydroxylation of the HIF-α subunit. It is proposed that the activity of the HIF hydroxylases can be regulated by their reaction with nitric oxide. We describe biochemical and biophysical studies on the reaction of prolyl hydroxylase domain-containing enzyme (PHD) isoform 2 (EGLN1) with nitric oxide and a nitric oxide transfer reagent. The combined results reveal the potential for the catalytic domain of PHD2 to react with nitric oxide both at its Fe(II) and at cysteine residues. Although the biological significance is unclear, the results suggest that the reaction of PHD2 with nitric oxide has the potential to be complex and are consistent with proposals based on cellular studies that nitric oxide may regulate the hypoxic response by direct reaction with the HIF hydroxylases.
|
Jul 2011
|
|
