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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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I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: De novo protein design is advancing rapidly. However, most designs are for single states. Here we report a de novo designed peptide that forms multiple α-helical-bundle states that are accessible and interconvertible under the same conditions. Usually in such designs amphipathic α helices associate to form compact structures with consolidated hydrophobic cores. However, recent rational and computational designs have delivered open α-helical barrels with functionalisable cavities. By placing glycine judiciously in the helical interfaces of an α-helical barrel, we obtain both open and compact states in a single protein crystal. Molecular dynamics simulations indicate a free-energy landscape with multiple and interconverting states. Together, these findings suggest a frustrated system in which steric interactions that maintain the open barrel and the hydrophobic effect that drives complete collapse are traded-off. Indeed, addition of a hydrophobic co-solvent that can bind within the barrel affects the switch between the states both in silico and experimentally.
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Mar 2021
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[12633]
Abstract: We report an engineered panel of ene-reductases (ERs) from Thermus scotoductus SA-01 (TsER) that combines control over facial selectivity in the reduction of electron deficient Cdouble bondC double bonds with thermostability (up to 70 °C), organic solvent tolerance (up to 40 % v/v) and a broad substrate scope (23 compounds, three new to literature). Substrate acceptance and facial selectivity of 3-methylcyclohexenone was rationalized by crystallisation of TsER C25D/I67T and in silico docking. The TsER variant panel shows excellent enantiomeric excess (ee) and yields during bi-phasic preparative scale synthesis, with isolated yield of up to 93 % for 2R,5S-dihydrocarvone (3.6 g). Turnover frequencies (TOF) of approximately 40 000 h−1 were achieved, which are comparable to rates in hetero- and homogeneous metal catalysed hydrogenations. Preliminary batch reactions also demonstrated the reusability of the reaction system by consecutively removing the organic phase (n-pentane) for product removal and replacing with fresh substrate. Four consecutive batches yielded ca. 27 g L−1 R-levodione from a 45 mL aqueous reaction, containing less than 17 mg (10 μM) enzyme and the reaction only stopping because of acidification. The TsER variant panel provides a robust, highly active and stereocomplementary base for further exploitation as a tool in preparative organic synthesis.
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Feb 2021
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[172122, 23269]
Open Access
Abstract: Class A serine β-lactamases (SBLs) are key antibiotic resistance determinants in Gram-negative bacteria. SBLs neutralize β-lactams via a hydrolytically labile covalent acyl-enzyme intermediate. Klebsiella pneumoniae carbapenemase (KPC) is a widespread SBL that hydrolyzes carbapenems, the most potent β-lactams; known KPC variants differ in turnover of expanded-spectrum oxyimino-cephalosporins (ESOCs), e.g. cefotaxime and ceftazidime. Here, we compare ESOC hydrolysis by the parent enzyme KPC-2 and its clinically observed double variant (P104R/V240G) KPC-4. Kinetic analyses show KPC-2 hydrolyzes cefotaxime more efficiently than the bulkier ceftazidime, with improved ESOC turnover by KPC-4 resulting from enhanced turnover (kcat), rather than binding (KM). High-resolution crystal structures of ESOC acyl-enzyme complexes with deacylation-deficient (E166Q) KPC-2 and KPC-4 mutants show that ceftazidime acylation causes rearrangement of three loops; the Ω-, 240- and 270-loops, that border the active site. However, these rearrangements are less pronounced in the KPC-4 than the KPC-2 ceftazidime acyl-enzyme, and are not observed in the KPC-2:cefotaxime acyl-enzyme. Molecular dynamics simulations of KPC:ceftazidime acyl-enyzmes reveal that the deacylation general base E166, located on the Ω-loop, adopts two distinct conformations in KPC-2, either pointing ‘in’ or ‘out’ of the active site; with only the ‘in’ form compatible with deacylation. The ‘out’ conformation was not sampled in the KPC-4 acyl-enzyme, indicating that efficient ESOC breakdown is dependent upon the ordering and conformation of the KPC Ω-loop. The results explain how point mutations expand the activity spectrum of the clinically important KPC SBLs to include ESOCs through their effects on the conformational dynamics of the acyl-enzyme intermediate.
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Nov 2020
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I24-Microfocus Macromolecular Crystallography
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Emily
Lythell
,
Reynier
Suardíaz
,
Philip
Hinchliffe
,
Chonnikan
Hanpaibool
,
Surawit
Visitsatthawong
,
A. Sofia F.
Oliveira
,
Eric J. M.
Lang
,
Panida
Surawatanawong
,
Vannajan Sanghiran
Lee
,
Thanyada
Rungrotmongkol
,
Natalie
Fey
,
James
Spencer
,
Adrian J.
Mulholland
Diamond Proposal Number(s):
[12342]
Abstract: MCR (mobile colistin resistance) enzymes catalyse phosphoethanolamine (PEA) addition to bacterial lipid A, threatening the “last-resort” antibiotic colistin. Molecular dynamics and density functional theory simulations indicate that monozinc MCR supports PEA transfer to the Thr285 acceptor, positioning MCR as a mono- rather than multinuclear member of the alkaline phosphatase superfamily.
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May 2020
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