I03-Macromolecular Crystallography
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Open Access
Abstract: The essential L,D-transpeptidase of Mycobacterium tuberculosis (LdtMt2) catalyses the formation of 33 cross-links in cell wall peptidoglycan and is a target for development of antituberculosis therapeutics. Efforts to inhibit LdtMt2 have been hampered by lack of knowledge of how it binds its substrate. To address this gap, we optimised the isolation of natural disaccharide tetrapeptide monomers from the Corynebacterium jeikeium bacterial cell wall through overproduction of the peptidoglycan sacculus. The tetrapeptides were used in binding / turnover assays and biophysical studies on LdtMt2. We determined a crystal structure of wild-type LdtMt2 reacted with its natural substrate, the tetrapeptide monomer of the peptidoglycan layer. This structure shows formation of a thioester linking the catalytic cysteine and the donor substrate, reflecting an intermediate in the transpeptidase reaction; it informs on the mode of entrance of the donor substrate into the LdtMt2 active site. The results will be useful in design of LdtMt2 inhibitors, including those based on substrate binding interactions, a strategy successfully employed for other nucleophilic cysteine enzymes.
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Sep 2024
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I03-Macromolecular Crystallography
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Open Access
Abstract: Disruption of bacterial cell wall biosynthesis in Mycobacterium tuberculosis is a promising target for treating tuberculosis. The L,D-transpeptidase LdtMt2, which is responsible for the formation of 3□(→)3 cross-links in the cell wall peptidoglycan, has been identified as essential for M. tuberculosis virulence. We optimised a high-throughput assay for LdtMt2, and screened a targeted library of ~10.000 electrophilic compounds. Potent inhibitor classes were identified, including established (e.g. β-lactams) and unexplored covalently reacting electrophilic groups (e.g., cyanamides). Protein-observed mass spectrometric studies reveal most classes to react covalently and irreversibly with the LdtMt2 catalytic cysteine (Cys354). Crystallographic analyses of seven representative inhibitors reveal induced fit involving a loop enclosing the LdtMt2 active site. Several of the identified compounds have a bactericidal effect on M. tuberculosis within macrophages, one with an MIC50 value of ~1 µM. The results provide leads for the development of new covalently reaction inhibitors of LdtMt2 and other nucleophilic cysteine enzymes.
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May 2023
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19458, 23459]
Open Access
Abstract: β-Lactams are the most important class of antibacterials, but their use is increasingly compromised by resistance, most importantly via serine β-lactamase (SBL)-catalyzed hydrolysis. The scope of β-lactam antibacterial activity can be substantially extended by coadministration with a penicillin-derived SBL inhibitor (SBLi), i.e., the penam sulfones tazobactam and sulbactam, which are mechanism-based inhibitors working by acylation of the nucleophilic serine. The new SBLi enmetazobactam, an N-methylated tazobactam derivative, has recently completed clinical trials. Biophysical studies on the mechanism of SBL inhibition by enmetazobactam reveal that it inhibits representatives of all SBL classes without undergoing substantial scaffold fragmentation, a finding that contrasts with previous reports on SBL inhibition by tazobactam and sulbactam. We therefore reinvestigated the mechanisms of tazobactam and sulbactam using mass spectrometry under denaturing and nondenaturing conditions, X-ray crystallography, and NMR spectroscopy. The results imply that the reported extensive fragmentation of penam sulfone–derived acyl–enzyme complexes does not substantially contribute to SBL inhibition. In addition to observation of previously identified inhibitor-induced SBL modifications, the results reveal that prolonged reaction of penam sulfones with SBLs can induce dehydration of the nucleophilic serine to give a dehydroalanine residue that undergoes reaction to give a previously unobserved lysinoalanine cross-link. The results clarify the mechanisms of action of widely clinically used SBLi, reveal limitations on the interpretation of mass spectrometry studies concerning mechanisms of SBLi, and will inform the development of new SBLi working by reaction to form hydrolytically stable acyl–enzyme complexes.
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May 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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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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I24-Microfocus Macromolecular Crystallography
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Agata
Butryn
,
Philipp S.
Simon
,
Pierre
Aller
,
Philip
Hinchliffe
,
Ramzi N.
Massad
,
Gabriel
Leen
,
Catherine L.
Tooke
,
Isabel
Bogacz
,
In-Sik
Kim
,
Asmit
Bhowmick
,
Aaron S.
Brewster
,
Nicholas E.
Devenish
,
Jurgen
Brem
,
Jos J. A. G.
Kamps
,
Pauline A.
Lang
,
Patrick
Rabe
,
Danny
Axford
,
John H.
Beale
,
Bradley
Davy
,
Ali
Ebrahim
,
Julien
Orlans
,
Selina L. S.
Storm
,
Tiankun
Zhou
,
Shigeki
Owada
,
Rie
Tanaka
,
Kensuke
Tono
,
Gwyndaf
Evans
,
Robin L.
Owen
,
Frances A.
Houle
,
Nicholas K.
Sauter
,
Christopher J.
Schofield
,
James
Spencer
,
Vittal K.
Yachandra
,
Junko
Yano
,
Jan F.
Kern
,
Allen M.
Orville
Diamond Proposal Number(s):
[19458, 25260]
Open Access
Abstract: Serial femtosecond crystallography has opened up many new opportunities in structural biology. In recent years, several approaches employing light-inducible systems have emerged to enable time-resolved experiments that reveal protein dynamics at high atomic and temporal resolutions. However, very few enzymes are light-dependent, whereas macromolecules requiring ligand diffusion into an active site are ubiquitous. In this work we present a drop-on-drop sample delivery system that enables the study of enzyme-catalyzed reactions in microcrystal slurries. The system delivers ligand solutions in bursts of multiple picoliter-sized drops on top of a larger crystal-containing drop inducing turbulent mixing and transports the mixture to the X-ray interaction region with temporal resolution. We demonstrate mixing using fluorescent dyes, numerical simulations and time-resolved serial femtosecond crystallography, which show rapid ligand diffusion through microdroplets. The drop-on-drop method has the potential to be widely applicable to serial crystallography studies, particularly of enzyme reactions with small molecule substrates.
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Jul 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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I03-Macromolecular Crystallography
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Emma Zsófia Aletta
Nagy
,
Souad Diana
Tork
,
Pauline A.
Lang
,
Alina
Filip
,
Florin Dan
Irimie
,
László
Poppe
,
Monica Ioana
Toşa
,
Christopher J.
Schofield
,
Jurgen
Brem
,
Csaba
Paizs
,
Laszlo Csaba
Bencze
Diamond Proposal Number(s):
[19458]
Abstract: Modification of the hydrophobic binding pocket of phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) enables increased activity and selectivity towards phenylalanines and cinnamic acids mono-substituted with both electron donating (-CH3, -OCH3) and electron withdrawing (-CF3, -Br) groups at all positions (o-, m-, p-) of their aromatic ring. The results reveal specific residues involved in accommodating substituents at o-, m-, p-positions of the substrate’s phenyl ring. The predicted interactions were validated by crystallographic analysis of the binding mode of para-methoxy cinnamic acid complexed at the active site of PcPAL. The biocatalytic utility of the tailored PcPAL mutants was demonstrated by the efficient preparative scale synthesis of (S)-m-bromo-phenylalanine (ee: > 99%, yield: 60%) and (R)-p-methyl-phenylalanine (ee: 97%, yield: 49%), using the corresponding ammonia addition and ammonia elimination reactions catalyzed by the L134A and I460V PcPAL variants, respectively. Overall, the results reveal the potential for structure based protein engineering of PALs to provide enzymes with enhanced catalytic properties and which are specifically tailored for differently substituted phenylalanine analogues of high synthetic value.
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Aug 2019
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