I03-Macromolecular Crystallography
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Michael
Beer
,
Philip
Hinchliffe
,
Marko
Hanževački
,
Christopher R.
Bethel
,
Catherine L.
Tooke
,
Marc W.
Van Der Kamp
,
Krisztina M.
Papp‐wallace
,
Robert A.
Bonomo
,
Stuart
Shapiro
,
Adrian J.
Mulholland
,
James
Spencer
Diamond Proposal Number(s):
[23269, 31440]
Open Access
Abstract: β-Lactamase-catalysed hydrolysis is the primary form of β-lactam antibiotic resistance in Gram-negative bacteria. The penicillanic acid sulfone (PAS) enmetazobactam is thought to inhibit extended-spectrum β-lactamases (ESBLs) by fragmentation of an initial acyl-enzyme to form an active-site lysinoalanine cross link. We investigate interactions of enmetazobactam and its congener tazobactam with GES-1, an ESBL with structural features of carbapenem-hydrolysing β-lactamases. Crystal structures show different breakdown products of the two inhibitors covalently bound to the catalytic Ser70, assigned using quantum mechanics/molecular mechanics (QM/MM) calculations. We find no evidence for lysinoalanine formation, with mass spectrometry indicating active enzyme regeneration, behaviour previously observed for carbapenem-hydrolysing enzymes, but not ESBLs. This work establishes that PAS inhibitors interact with diverse β-lactamases by differing mechanisms, which should inform development of future compounds.
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Sep 2025
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I24-Microfocus Macromolecular Crystallography
VMXi-Versatile Macromolecular Crystallography in situ
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Hans E.
Pfalzgraf
,
Aditya G.
Rao
,
Kakali
Sen
,
Hannah R.
Adams
,
Marcus
Edwards
,
You
Lu
,
Chin
Yong
,
Sofia
Jaho
,
Takehiko
Tosha
,
Hiroshi
Sugimoto
,
Sam
Horrell
,
James
Beilsten-Edmands
,
Robin L.
Owen
,
Colin R.
Andrew
,
Jonathan A. R.
Worrall
,
Ivo
Tews
,
Adrian J.
Mulholland
,
Michael A.
Hough
,
Thomas W.
Keal
Diamond Proposal Number(s):
[27313]
Open Access
Abstract: Cytochromes P460 oxidise hydroxylamine within the nitrogen cycle and contain as their active site an unusual catalytic c-type haem where the porphyrin is crosslinked to the protein via a lysine residue in addition to the canonical cross links from cysteine residues. Understanding how enzymes containing P460 haem oxidise hydroxylamine into either nitrous oxide or nitric oxide has implications for climate change. Interestingly the P460-containing hydroxylamine oxidoreductase utilises a tyrosine cross link to haem and performs similar chemistry. Previous crystal structures of cytochrome P460 from Nitrosomonas europaea (NeP460) clearly show the existence of a single crosslink between the NZ atom of lysine and the haem porphyrin, with mutagenesis studies indicating roles for the crosslink in positioning a proton transfer residue and/or influencing the distortion of the haem. Here we describe the evidence for a novel double crosslink between lysine and haem in the cytochrome P460 from Methylococcus capsulatus (Bath). In order to understand the complexities of this enzyme system we applied high resolution structural biology approaches at synchrotron and XFEL sources paired with crystal spectroscopies. Linked to this, we carried out QM/MM simulations that enabled the prediction of electronic absorption spectra providing a crucial validation to linking simulations and experimental structures. Our work demonstrates the feasibility of a double crosslink in McP460 and provides an opportunity to investigate how simulations can interact with experimental structures.
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Aug 2025
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Joseph F.
Hoff
,
Kirsty E.
Goudar
,
Karina
Calvopina
,
Michael
Beer
,
Philip
Hinchliffe
,
John M.
Shaw
,
Catherine L.
Tooke
,
Yuiko
Takebayashi
,
Andrew F.
Cadzow
,
Nicholas
Harmer
,
Adrian J.
Mulholland
,
Christopher J.
Schofield
,
James
Spencer
Diamond Proposal Number(s):
[23269, 31440]
Open Access
Abstract: Carbapenemases, β-lactamases hydrolysing carbapenem antibiotics, challenge treatment of multi-drug resistant bacteria. The OXA-48 carbapenemase is widely disseminated in Enterobacterales, necessitating new treatments for producer strains. Diazabicyclooctane (DBO) inhibitors, including avibactam and nacubactam, act on a wide range of enzymes to overcome β-lactamase-mediated resistance. Here we investigate avibactam and nacubactam activities towards OXA-48 and two variants, OXA-163 and OXA-405, with deletions in the β5 – β6 loop neighbouring the active site that modify activity towards different β-lactam classes. Nacubactam is c. 80-fold less potent than avibactam towards OXA-48, but this difference reduces in OXA-163 and OXA-405. Crystal structures and molecular dynamics simulations reveal electrostatic repulsion between Arg214 on the OXA-48 β5 – β6 active-site loop and nacubactam, but not avibactam, effects absent from simulations of OXA-163 and OXA-405, which lack Arg214. Crystallographic and mass spectrometry data demonstrate that all three enzymes support desulfation of bound DBOs. These data indicate that interactions with Arg214 affect DBO potency, suggesting that sequence variation in OXA-48-like β-lactamases affects reactivity towards inhibitors as well as β-lactam substrates.
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Aug 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[23269, 31440]
Open Access
Abstract: β-Lactamases, which hydrolyse β-lactam antibiotics, are key determinants of antibiotic resistance. Predicting the sites and effects of distal mutations in enzymes is challenging. For β-lactamases, the ability to make such predictions would contribute to understanding activity against, and development of, antibiotics and inhibitors to combat resistance. Here, using dynamical non-equilibrium molecular dynamics (D-NEMD) simulations combined with experiments, we demonstrate that intramolecular communication networks differ in three class A SulpHydryl Variant (SHV)-type β-lactamases. Differences in network architecture and correlated motions link to catalytic efficiency and β-lactam substrate spectrum. Further, the simulations identify a distal residue at position 89 in the clinically important Klebsiella pneumoniae carbapenemase 2 (KPC-2), as a participant in similar networks, suggesting that mutation at this position would modulate enzyme activity. Experimental kinetic, biophysical and structural characterisation of the naturally occurring, but previously biochemically uncharacterised, KPC-2G89D mutant with several antibiotics and inhibitors reveals significant changes in hydrolytic spectrum, specifically reducing activity towards carbapenems without effecting major structural or stability changes. These results show that D-NEMD simulations can predict distal sites where mutation affects enzyme activity. This approach could have broad application in understanding enzyme evolution, and in engineering of natural and de novo enzymes.
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Sep 2024
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Open Access
Abstract: The diffusion in ZSM-5 zeolite of methanol and of two series of promoters of the methanol to dimethyl ether reaction (linear methyl esters, benzaldehyde, 4-n-alkyl benzaldehydes) has been studied using classical molecular dynamics in the NVT ensemble. Whereas promoter diffusion coefficients decrease with increasing alkyl chain length in methyl esters, the aromatic aldehyde promoters all have similar diffusion coefficients. The lowest diffusion coefficient is that of benzaldehyde. All the promoters exhibit a preference for moving in the straight pore, a preference that is most pronounced for the 4-n-alkylbenzaldehydes and least for the longest aliphatic esters. A novel diffusion mechanism, a molecular ‘3-point turn’, is observed. This likely plays an important role in allowing the most potent promoters, with longer linear alkyl chains, to access all of the Brønsted acid reaction sites. The diffusion coefficient of methanol is larger than that of all the promoters. The more catalytically active aromatic aldehyde promoters limit methanol diffusion less than the aliphatic esters.
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May 2024
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I03-Macromolecular Crystallography
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George
Hutchins
,
Claire E. M.
Noble
,
H. Adrian
Bunzel
,
Christopher
Williams
,
Paulina
Dubiel
,
Sathish K. N.
Yadav
,
Paul M.
Molinaro
,
Rob
Barringer
,
Hector
Blackburn
,
Benjamin
Hardy
,
Alice E.
Parnell
,
Charles
Landau
,
Paul R.
Race
,
Thomas A. A.
Oliver
,
Ronald L.
Koder
,
Matthew P.
Crump
,
Christiane
Schaffitzel
,
A. Sofia F.
Oliveira
,
Adrian J.
Mulholland
,
J. L. Ross
Anderson
Diamond Proposal Number(s):
[31440]
Open Access
Abstract: The electron-conducting circuitry of life represents an as-yet untapped resource of exquisite, nanoscale biomolecular engineering. Here, we report the characterization and structure of a de novo diheme “maquette” protein, 4D2, which we subsequently use to create an expanded, modular platform for heme protein design. A well-folded monoheme variant was created by computational redesign, which was then utilized for the experimental validation of continuum electrostatic redox potential calculations. This demonstrates how fundamental biophysical properties can be predicted and fine-tuned. 4D2 was then extended into a tetraheme helical bundle, representing a 7 nm molecular wire. Despite a molecular weight of only 24 kDa, electron cryomicroscopy illustrated a remarkable level of detail, indicating the positioning of the secondary structure and the heme cofactors. This robust, expressible, highly thermostable and readily designable modular platform presents a valuable resource for redox protein design and the future construction of artificial electron-conducting circuitry.
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Aug 2023
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[172122, 23269]
Open Access
Abstract: KPC-2 (Klebsiella pneumoniae carbapenemase-2) is a globally disseminated serine-β-lactamase (SBL) responsible for extensive β-lactam antibiotic resistance in Gram-negative pathogens. SBLs inactivate β-lactams via a mechanism involving a hydrolytically labile covalent acyl-enzyme intermediate. Carbapenems, the most potent β-lactams, evade the activity of many SBLs by forming long-lived inhibitory acyl-enzymes; however, carbapenemases such as KPC-2 efficiently deacylate carbapenem acyl-enzymes. We present high-resolution (1.25–1.4 Å) crystal structures of KPC-2 acyl-enzymes with representative penicillins (ampicillin), cephalosporins (cefalothin), and carbapenems (imipenem, meropenem, and ertapenem) obtained utilizing an isosteric deacylation-deficient mutant (E166Q). The mobility of the Ω-loop (residues 165–170) negatively correlates with antibiotic turnover rates (kcat), highlighting the role of this region in positioning catalytic residues for efficient hydrolysis of different β-lactams. Carbapenem-derived acyl-enzyme structures reveal the predominance of the Δ1-(2R) imine rather than the Δ2 enamine tautomer. Quantum mechanics/molecular mechanics molecular dynamics simulations of KPC-2:meropenem acyl-enzyme deacylation used an adaptive string method to differentiate the reactivity of the two isomers. These identify the Δ1-(2R) isomer as having a significantly (7 kcal/mol) higher barrier than the Δ2 tautomer for the (rate-determining) formation of the tetrahedral deacylation intermediate. Deacylation is therefore likely to proceed predominantly from the Δ2, rather than the Δ1-(2R) acyl-enzyme, facilitated by tautomer-specific differences in hydrogen-bonding networks involving the carbapenem C-3 carboxylate and the deacylating water and stabilization by protonated N-4, accumulating a negative charge on the Δ2 enamine-derived oxyanion. Taken together, our data show how the flexible Ω-loop helps confer broad-spectrum activity upon KPC-2, while carbapenemase activity stems from efficient deacylation of the Δ2-enamine acyl-enzyme tautomer.
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Mar 2023
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[17212]
Abstract: Widespread bacterial resistance to carbapenem antibiotics is an increasing global health concern. Resistance has emerged due to carbapenem-hydrolyzing enzymes, including metallo-β-lactamases (MβLs), but despite their prevalence and clinical importance, MβL mechanisms are still not fully understood. Carbapenem hydrolysis by MβLs can yield alternative product tautomers with the potential to access different binding modes. Here, we show that a combined approach employing crystallography and quantum mechanics/molecular mechanics (QM/MM) simulations allow tautomer assignment in MβL:hydrolyzed antibiotic complexes. Molecular simulations also examine (meta)stable species of alternative protonation and tautomeric states, providing mechanistic insights into β-lactam hydrolysis. We report the crystal structure of the hydrolyzed carbapenem ertapenem bound to the L1 MβL from Stenotrophomonas maltophilia and model alternative tautomeric and protonation states of both hydrolyzed ertapenem and faropenem (a related penem antibiotic), which display different binding modes with L1. We show how the structures of both complexed β-lactams are best described as the (2S)-imine tautomer with the carboxylate formed after β-lactam ring cleavage deprotonated. Simulations show that enamine tautomer complexes are significantly less stable (e.g., showing partial loss of interactions with the L1 binuclear zinc center) and not consistent with experimental data. Strong interactions of Tyr32 and one zinc ion (Zn1) with ertapenem prevent a C6 group rotation, explaining the different binding modes of the two β-lactams. Our findings establish the relative stability of different hydrolyzed (carba)penem forms in the L1 active site and identify interactions important to stable complex formation, information that should assist inhibitor design for this important antibiotic resistance determinant.
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Oct 2021
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NONE-No attached Diamond beamline
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H. T. Henry
Chan
,
Marc A.
Moesser
,
Rebecca K.
Walters
,
Tika R.
Malla
,
Rebecca M.
Twidale
,
Tobias
John
,
Helen M.
Deeks
,
Tristan
Johnston-Wood
,
Victor
Mikhailov
,
Richard B.
Sessions
,
William
Dawson
,
Eidarus
Salah
,
Petra
Lukacik
,
Claire
Strain-Damerell
,
C. David
Owen
,
Takahito
Nakajima
,
Katarzyna
Świderek
,
Alessio
Lodola
,
Vicent
Moliner
,
David R.
Glowacki
,
James
Spencer
,
Martin A.
Walsh
,
Christopher J.
Schofield
,
Luigi
Genovese
,
Deborah K.
Shoemark
,
Adrian J.
Mulholland
,
Fernanda
Duarte
,
Garrett M.
Morris
Open Access
Abstract: The main protease (Mpro) of SARS-CoV-2 is central to viral maturation and is a promising drug target, but little is known about structural aspects of how it binds to its 11 natural cleavage sites. We used biophysical and crystallographic data and an array of biomolecular simulation techniques, including automated docking, molecular dynamics (MD) and interactive MD in virtual reality, QM/MM, and linear-scaling DFT, to investigate the molecular features underlying recognition of the natural Mpro substrates. We extensively analysed the subsite interactions of modelled 11-residue cleavage site peptides, crystallographic ligands, and docked COVID Moonshot-designed covalent inhibitors. Our modelling studies reveal remarkable consistency in the hydrogen bonding patterns of the natural Mpro substrates, particularly on the N-terminal side of the scissile bond. They highlight the critical role of interactions beyond the immediate active site in recognition and catalysis, in particular plasticity at the S2 site. Building on our initial Mpro-substrate models, we used predictive saturation variation scanning (PreSaVS) to design peptides with improved affinity. Non-denaturing mass spectrometry and other biophysical analyses confirm these new and effective ‘peptibitors’ inhibit Mpro competitively. Our combined results provide new insights and highlight opportunities for the development of Mpro inhibitors as anti-COVID-19 drugs.
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Oct 2021
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Alistair J.
Scott
,
Ai
Niitsu
,
Huong T.
Kratochvil
,
Eric J. M.
Lang
,
Jason T.
Sengel
,
William M.
Dawson
,
Kozhinjampara R.
Mahendran
,
Marco
Mravic
,
Andrew
Thomson
,
R. Leo
Brady
,
Lijun
Liu
,
Adrian J.
Mulholland
,
Hagan
Bayley
,
William F.
Degrado
,
Mark I.
Wallace
,
Derek N.
Woolfson
Abstract: The design of peptides that assemble in membranes to form functional ion channels is challenging. Specifically, hydrophobic interactions must be designed between the peptides and at the peptide–lipid interfaces simultaneously. Here, we take a multi-step approach towards this problem. First, we use rational de novo design to generate water-soluble α-helical barrels with polar interiors, and confirm their structures using high-resolution X-ray crystallography. These α-helical barrels have water-filled lumens like those of transmembrane channels. Next, we modify the sequences to facilitate their insertion into lipid bilayers. Single-channel electrical recordings and fluorescent imaging of the peptides in membranes show monodisperse, cation-selective channels of unitary conductance. Surprisingly, however, an X-ray structure solved from the lipidic cubic phase for one peptide reveals an alternative state with tightly packed helices and a constricted channel. To reconcile these observations, we perform computational analyses to compare the properties of possible different states of the peptide.
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May 2021
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