I03-Macromolecular Crystallography
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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.
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Nov 2022
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Jurgen
Brem
,
Tharindi
Panduwawala
,
Jon Ulf
Hansen
,
Joanne
Hewitt
,
Edgars
Liepins
,
Pawel
Donets
,
Laura
Espina
,
Alistair J. M.
Farley
,
Kirill
Shubin
,
Gonzalo Gomez
Campillos
,
Paula
Kiuru
,
Shifali
Shishodia
,
Daniel
Krahn
,
Robert K.
Leśniak
,
Juliane
Schmidt
,
Karina
Calvopina
,
María-Carmen
Turrientes
,
Madeline E.
Kavanagh
,
Dmitrijs
Lubriks
,
Philip
Hinchliffe
,
Gareth W.
Langley
,
Ali F.
Aboklaish
,
Anders
Eneroth
,
Maria
Backlund
,
Andrei G.
Baran
,
Elisabet I.
Nielsen
,
Michael
Speake
,
Janis
Kuka
,
John
Robinson
,
Solveiga
Grinberga
,
Lindsay
Robinson
,
Michael A.
Mcdonough
,
Anna M.
Rydzik
,
Thomas M.
Leissing
,
Juan Carlos
Jimenez-Castellanos
,
Matthew B.
Avison
,
Solange
Da Silva Pinto
,
Andrew D.
Pannifer
,
Marina
Martjuga
,
Emma
Widlake
,
Martins
Priede
,
Iva
Hopkins Navratilova
,
Marek
Gniadkowski
,
Anna Karin
Belfrage
,
Peter
Brandt
,
Jari
Yli-Kauhaluoma
,
Eric
Bacque
,
Malcolm G. P.
Page
,
Fredrik
Björkling
,
Jonathan M.
Tyrrell
,
James
Spencer
,
Pauline A.
Lang
,
Pawel
Baranczewski
,
Rafael
Cantón
,
Stuart P.
Mcelroy
,
Philip S.
Jones
,
Fernando
Baquero
,
Edgars
Suna
,
Angus
Morrison
,
Timothy R.
Walsh
,
Christopher J.
Schofield
Open Access
Abstract: Carbapenems are vital antibiotics, but their efficacy is increasingly compromised by metallo-β-lactamases (MBLs). Here we report the discovery and optimization of potent broad-spectrum MBL inhibitors. A high-throughput screen for NDM-1 inhibitors identified indole-2-carboxylates (InCs) as potential β-lactamase stable β-lactam mimics. Subsequent structure–activity relationship studies revealed InCs as a new class of potent MBL inhibitor, active against all MBL classes of major clinical relevance. Crystallographic studies revealed a binding mode of the InCs to MBLs that, in some regards, mimics that predicted for intact carbapenems, including with respect to maintenance of the Zn(II)-bound hydroxyl, and in other regards mimics binding observed in MBL–carbapenem product complexes. InCs restore carbapenem activity against multiple drug-resistant Gram-negative bacteria and have a low frequency of resistance. InCs also have a good in vivo safety profile, and when combined with meropenem show a strong in vivo efficacy in peritonitis and thigh mouse infection models.
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Dec 2021
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Shifali
Shishodia
,
Marina
Demetriades
,
Dong
Zhang
,
Nok Yin
Tam
,
Pratheesh
Maheswaran
,
Caitlin
Clunie-O'Connor
,
Anthony
Tumber
,
Ivanhoe K. H.
Leung
,
Yi Min
Ng
,
Thomas M.
Leissing
,
Afaf H.
El-Sagheer
,
Eidarus
Salah
,
Tom
Brown
,
Wei Shen
Aik
,
Michael A.
Mcdonough
,
Christopher J.
Schofield
Open Access
Abstract: FTO catalyzes the Fe(II) and 2-oxoglutarate (2OG)-dependent modification of nucleic acids, including the demethylation of N6-methyladenosine (m6A) in mRNA. FTO is a proposed target for anti-cancer therapy. Using information from crystal structures of FTO in complex with 2OG and substrate mimics, we designed and synthesized two series of FTO inhibitors, which were characterized by turnover and binding assays, and by X-ray crystallography with FTO and the related bacterial enzyme AlkB. A potent inhibitor employing binding interactions spanning the FTO 2OG and substrate binding sites was identified. Selectivity over other clinically targeted 2OG oxygenases was demonstrated, including with respect to the hypoxia-inducible factor prolyl and asparaginyl hydroxylases (PHD2 and FIH) and selected JmjC histone demethylases (KDMs). The results illustrate how structure-based design can enable the identification of potent and selective 2OG oxygenase inhibitors and will be useful for the development of FTO inhibitors for use in vivo.
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Nov 2021
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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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.
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Aug 2021
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19069, 19458]
Open Access
Abstract: β-Lactam antibiotics are presently the most important treatments for infections by pathogenic Escherichia coli, but their use is increasingly compromised by β-lactamases, including the chromosomally encoded class C AmpC serine-β-lactamases (SBL). The diazabicyclooctane (DBO) avibactam is a potent AmpC inhibitor; the clinical success of avibactam combined with ceftazidime has stimulated efforts to optimise the DBO core. We report kinetic and structural studies, including four high resolution crystal structures, concerning inhibition of the AmpC serine-β-lactamase from E. coli (AmpCEC) by clinically relevant DBO-based inhibitors: avibactam, relebactam, nacubactam, and zidebactam. Kinetic analyses and mass spectrometry-based assays were used to study their mechanisms of AmpCEC inhibition. The results reveal that, under our assay conditions, zidebactam manifests increased potency (Kiapp 0.69 μM) against AmpCEC compared to the other DBOs (Kiapp 5.0-7.4 μM) due to an ∼ 10 fold accelerated carbamoylation-rate. However, zidebactam also has an accelerated off-rate and with sufficient preincubation time all the DBOs manifest similar potencies. Crystallographic analyses indicate a greater conformational freedom of the AmpCEC-zidebactam carbamoyl-complex compared to those for the other DBOs. The results suggest carbamoyl-complex lifetime should be a consideration in development of DBO-based SBL inhibitors for the clinically important class C SBLs.
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Nov 2020
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I24-Microfocus Macromolecular Crystallography
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Patrick
Rabe
,
John
Beale
,
Agata
Butryn
,
Pierre
Aller
,
Anna
Dirr
,
Pauline A.
Lang
,
Danny N.
Axford
,
Stephen
Carr
,
Thomas M.
Leissing
,
Michael A.
Mcdonough
,
Bradley
Davy
,
Ali
Ebrahim
,
Julien
Orlans
,
Selina L. S.
Storm
,
Allen M.
Orville
,
Christopher J.
Schofield
,
Robin L.
Owen
Diamond Proposal Number(s):
[19458]
Open Access
Abstract: Cryogenic X-ray diffraction is a powerful tool for crystallographic studies on enzymes including oxygenases and oxidases. Amongst the benefits that cryo-conditions (usually employing a nitrogen cryo-stream at 100 K) enable, is data collection of dioxygen-sensitive samples. Although not strictly anaerobic, at low temperatures the vitreous ice conditions severely restrict O2 diffusion into and/or through the protein crystal. Cryo-conditions limit chemical reactivity, including reactions that require significant conformational changes. By contrast, data collection at room temperature imposes fewer restrictions on diffusion and reactivity; room-temperature serial methods are thus becoming common at synchrotrons and XFELs. However, maintaining an anaerobic environment for dioxygen-dependent enzymes has not been explored for serial room-temperature data collection at synchrotron light sources. This work describes a methodology that employs an adaptation of the `sheet-on-sheet' sample mount, which is suitable for the low-dose room-temperature data collection of anaerobic samples at synchrotron light sources. The method is characterized by easy sample preparation in an anaerobic glovebox, gentle handling of crystals, low sample consumption and preservation of a localized anaerobic environment over the timescale of the experiment (<5 min). The utility of the method is highlighted by studies with three X-ray-radiation-sensitive Fe(II)-containing model enzymes: the 2-oxoglutarate-dependent L-arginine hydroxylase VioC and the DNA repair enzyme AlkB, as well as the oxidase isopenicillin N synthase (IPNS), which is involved in the biosynthesis of all penicillin and cephalosporin antibiotics.
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Sep 2020
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Pauline
Lang
,
Anete
Parkova
,
Thomas
Leissing
,
Karina
Calvopina
,
Ricky
Cain
,
Alen
Krajnc
,
Tharindi
Panduwawala
,
Jules
Philippe
,
Colin W. G.
Fishwick
,
Peteris
Trapencieris
,
Malcolm
Page
,
Christopher J.
Schofield
,
Jurgen
Brem
Open Access
Abstract: Resistance to β-lactam antibacterials, importantly via production of β-lactamases, threatens their widespread use. Bicyclic boronates show promise as clinically useful, dual-action inhibitors of both serine- (SBL) and metallo- (MBL) β-lactamases. In combination with cefepime, the bicyclic boronate taniborbactam is in phase 3 clinical trials for treatment of complicated urinary tract infections. We report kinetic and crystallographic studies on the inhibition of AmpC, the class C β‑lactamase from Escherichia coli, by bicyclic boronates, including taniborbactam, with different C-3 side chains. The combined studies reveal that an acylamino side chain is not essential for potent AmpC inhibition by active site binding bicyclic boronates. The tricyclic form of taniborbactam was observed bound to the surface of crystalline AmpC, but not at the active site, where the bicyclic form was observed. Structural comparisons reveal insights into why active site binding of a tricyclic form has been observed with the NDM-1 MBL, but not with other studied β-lactamases. Together with reported studies on the structural basis of inhibition of class A, B and D β‑lactamases, our data support the proposal that bicyclic boronates are broad-spectrum β‑lactamase inhibitors that work by mimicking a high energy ‘tetrahedral’ intermediate. These results suggest further SAR guided development could improve the breadth of clinically useful β-lactamase inhibition.
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Jun 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Hwanho
Choi
,
Adam P.
Hardy
,
Thomas M.
Leissing
,
Rasheduzzaman
Chowdhury
,
Yu
Nakashima
,
Wei
Ge
,
Marios
Markoulides
,
John S.
Scotti
,
Philip A.
Gerken
,
Helen
Thorbjornsrud
,
Dahye
Kang
,
Sungwoo
Hong
,
Joongoo
Lee
,
Michael A.
Mcdonough
,
Hwangseo
Park
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[18069]
Open Access
Abstract: Factor inhibiting hypoxia-inducible factor (FIH) is a 2-oxoglutarate-dependent protein hydroxylase that catalyses C3 hydroxylations of protein residues. We report FIH can accept (D)- and (L)-residues for hydroxylation. The substrate selectivity of FIH differs for (D) and (L) epimers, e.g., (D)- but not (L)-allylglycine, and conversely (L)- but not (D)-aspartate, undergo monohydroxylation, in the tested sequence context. The (L)-Leu-containing substrate undergoes FIH-catalysed monohydroxylation, whereas (D)-Leu unexpectedly undergoes dihydroxylation. Crystallographic, mass spectrometric, and DFT studies provide insights into the selectivity of FIH towards (L)- and (D)-residues. The results of this work expand the potential range of known substrates hydroxylated by isolated FIH and imply that it will be possible to generate FIH variants with altered selectivities.
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May 2020
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