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Article ; Online: Room temperature crystallography of human acetylcholinesterase bound to a substrate analogue 4K-TMA

Oksana Gerlits / Matthew P. Blakeley / David A. Keen / Zoran Radić / Andrey Kovalevsky

Current Research in Structural Biology, Vol 3, Iss , Pp 206-

Towards a neutron structure

2021 Volume 215

Abstract: Acetylcholinesterase (AChE) catalyzes hydrolysis of acetylcholine thereby terminating cholinergic nerve impulses for efficient neurotransmission. Human AChE (hAChE) is a target of nerve agent and pesticide organophosphorus compounds that covalently ... ...

Abstract	Acetylcholinesterase (AChE) catalyzes hydrolysis of acetylcholine thereby terminating cholinergic nerve impulses for efficient neurotransmission. Human AChE (hAChE) is a target of nerve agent and pesticide organophosphorus compounds that covalently attach to the catalytic Ser203 residue. Reactivation of inhibited hAChE can be achieved with nucleophilic antidotes, such as oximes. Understanding structural and electrostatic (i.e. protonation states) determinants of the catalytic and reactivation processes is crucial to improve design of oxime reactivators. Here we report X-ray structures of hAChE conjugated with a reversible covalent inhibitor 4K-TMA (4K-TMA:hAChE) at 2.8 Å resolution and of 4K-TMA:hAChE conjugate with oxime reactivator methoxime, MMB4 (4K-TMA:hAChE:MMB4) at 2.6 Å resolution, both at physiologically relevant room temperature, as well as cryo-crystallographic structure of 4K-TMA:hAChE at 2.4 Å resolution. 4K-TMA acts as a substrate analogue reacting with the hydroxyl of Ser203 and generating a reversible tetrahedral hemiketal intermediate that closely resembles the first tetrahedral intermediate state during hAChE-catalyzed acetylcholine hydrolysis. Structural comparisons of room temperature with cryo-crystallographic structures of 4K-TMA:hAChE and published mAChE complexes with 4K-TMA, as well as the effect of MMB4 binding to the peripheral anionic site (PAS) of the 4K-TMA:hAChE complex, revealed only discrete, minor differences. The active center geometry of AChE, already highly evolved for the efficient catalysis, was thus indicative of only minor conformational adjustments to accommodate the tetrahedral intermediate in the hydrolysis of the neurotransmitter acetylcholine (ACh). To map protonation states in the hAChE active site gorge we collected 3.5 Å neutron diffraction data paving the way for obtaining higher resolution datasets that will be needed to determine locations of individual hydrogen atoms.
Keywords	X-ray diffraction ; Neutron diffraction ; hAChE ; Room temperature ; Reversible covalent inhibitor ; 4K-TMA ; Biology (General) ; QH301-705.5
Subject code	540
Language	English
Publishing date	2021-01-01T00:00:00Z
Publisher	Elsevier
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Neutron crystallography reveals mechanisms used by Pseudomonas aeruginosa for host-cell binding

Lukas Gajdos / Matthew P. Blakeley / Michael Haertlein / V. Trevor Forsyth / Juliette M. Devos / Anne Imberty

Nature Communications, Vol 13, Iss 1, Pp 1-

2022 Volume 9

Abstract: Pseudomonas aeruginosa employs lectins to bind to its host cells, and is known to be the major cause of lung infections. Lectin B (LecB) from Pseudomonas aeruginosa binds specifically to galactose and fucose and is important for pathogenicity, adhesion ... ...

Abstract	Pseudomonas aeruginosa employs lectins to bind to its host cells, and is known to be the major cause of lung infections. Lectin B (LecB) from Pseudomonas aeruginosa binds specifically to galactose and fucose and is important for pathogenicity, adhesion and biofilm formation. In this work, the neutron crystal structure (1.9 Å) of the deuterated LecB/Ca/fucose complex is reported. The structure, in combination with perdeuteration of the ligand and the receptor allowed the observation of hydrogen atoms, protonation states and hydrogen bonds involved in the interaction between pathogenic bacteria and host cells. Thus the study provides structural insights into the mechanism of high affinity binding of LecB to its targets.
Keywords	Science ; Q
Language	English
Publishing date	2022-01-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Joint neutron/X-ray crystal structure of a mechanistically relevant complex of perdeuterated urate oxidase and simulations provide insight into the hydration step of catalysis

Lindsay McGregor / Tamás Földes / Soi Bui / Martine Moulin / Nicolas Coquelle / Matthew P. Blakeley / Edina Rosta / Roberto A. Steiner

IUCrJ, Vol 8, Iss 1, Pp 46-

2021 Volume 59

Abstract: Cofactor-independent urate oxidase (UOX) is an ∼137 kDa tetrameric enzyme essential for uric acid (UA) catabolism in many organisms. UA is first oxidized by O2 to dehydroisourate (DHU) via a peroxo intermediate. DHU then undergoes hydration to 5- ... ...

Abstract	Cofactor-independent urate oxidase (UOX) is an ∼137 kDa tetrameric enzyme essential for uric acid (UA) catabolism in many organisms. UA is first oxidized by O2 to dehydroisourate (DHU) via a peroxo intermediate. DHU then undergoes hydration to 5-hydroxyisourate (5HIU). At different stages of the reaction both catalytic O2 and water occupy the `peroxo hole' above the organic substrate. Here, high-resolution neutron/X-ray crystallographic analysis at room temperature has been integrated with molecular dynamics simulations to investigate the hydration step of the reaction. The joint neutron/X-ray structure of perdeuterated Aspergillus flavus UOX in complex with its 8-azaxanthine (8AZA) inhibitor shows that the catalytic water molecule (W1) is present in the peroxo hole as neutral H2O, oriented at 45° with respect to the ligand. It is stabilized by Thr57 and Asn254 on different UOX protomers as well as by an O—H.π interaction with 8AZA. The active site Lys10–Thr57 dyad features a charged Lys10–NH3+ side chain engaged in a strong hydrogen bond with Thr57OG1, while the Thr57OG1–HG1 bond is rotationally dynamic and oriented toward the π system of the ligand, on average. Our analysis offers support for a mechanism in which W1 performs a nucleophilic attack on DHUC5 with Thr57HG1 central to a Lys10-assisted proton-relay system. Room-temperature crystallography and simulations also reveal conformational heterogeneity for Asn254 that modulates W1 stability in the peroxo hole. This is proposed to be an active mechanism to facilitate W1/O2 exchange during catalysis.
Keywords	neutron/x-ray diffraction ; urate oxidase ; protein perdeuteration ; cofactor-independent oxidase ; biomolecular simulations ; Science ; Q
Subject code	540
Language	English
Publishing date	2021-01-01T00:00:00Z
Publisher	International Union of Crystallography
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Neutron structures of Leishmania mexicana triosephosphate isomerase in complex with reaction-intermediate mimics shed light on the proton-shuttling steps

Vinardas Kelpšas / Octav Caldararu / Matthew P. Blakeley / Nicolas Coquelle / Rikkert K. Wierenga / Ulf Ryde / Claes von Wachenfeldt / Esko Oksanen

IUCrJ, Vol 8, Iss 4, Pp 633-

2021 Volume 643

Abstract: Triosephosphate isomerase (TIM) is a key enzyme in glycolysis that catalyses the interconversion of glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. This simple reaction involves the shuttling of protons mediated by protolysable side chains. ... ...

Abstract	Triosephosphate isomerase (TIM) is a key enzyme in glycolysis that catalyses the interconversion of glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. This simple reaction involves the shuttling of protons mediated by protolysable side chains. The catalytic power of TIM is thought to stem from its ability to facilitate the deprotonation of a carbon next to a carbonyl group to generate an enediolate intermediate. The enediolate intermediate is believed to be mimicked by the inhibitor 2-phosphoglycolate (PGA) and the subsequent enediol intermediate by phosphoglycolohydroxamate (PGH). Here, neutron structures of Leishmania mexicana TIM have been determined with both inhibitors, and joint neutron/X-ray refinement followed by quantum refinement has been performed. The structures show that in the PGA complex the postulated general base Glu167 is protonated, while in the PGH complex it remains deprotonated. The deuteron is clearly localized on Glu167 in the PGA–TIM structure, suggesting an asymmetric hydrogen bond instead of a low-barrier hydrogen bond. The full picture of the active-site protonation states allowed an investigation of the reaction mechanism using density-functional theory calculations.
Keywords	triosephosphate isomerase ; neutron diffraction ; isomerization ; quantum refinement ; qm/mm ; neutron crystallography ; refinement ; enzyme mechanisms ; structural biology ; Crystallography ; QD901-999
Subject code	540
Language	English
Publishing date	2021-07-01T00:00:00Z
Publisher	International Union of Crystallography
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Visualizing Tetrahedral Oxyanion Bound in HIV‑1 Protease Using Neutrons

Mukesh Kumar / Kalyaneswar Mandal / Matthew P. Blakeley / Troy Wymore / Stephen B. H. Kent / John M. Louis / Amit Das / Andrey Kovalevsky

ACS Omega, Vol 5, Iss 20, Pp 11605-

Implications for the Catalytic Mechanism and Drug Design

2020 Volume 11617

Keywords	Chemistry ; QD1-999
Language	English
Publishing date	2020-05-01T00:00:00Z
Publisher	American Chemical Society
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Sub-atomic resolution X-ray crystallography and neutron crystallography

Matthew P. Blakeley / Samar S. Hasnain / Svetlana V. Antonyuk

IUCrJ, Vol 2, Iss 4, Pp 464-

promise, challenges and potential

2015 Volume 474

Abstract: The International Year of Crystallography saw the number of macromolecular structures deposited in the Protein Data Bank cross the 100000 mark, with more than 90000 of these provided by X-ray crystallography. The number of X-ray structures determined to ... ...

Abstract	The International Year of Crystallography saw the number of macromolecular structures deposited in the Protein Data Bank cross the 100000 mark, with more than 90000 of these provided by X-ray crystallography. The number of X-ray structures determined to sub-atomic resolution (i.e. ≤1 Å) has passed 600 and this is likely to continue to grow rapidly with diffraction-limited synchrotron radiation sources such as MAX-IV (Sweden) and Sirius (Brazil) under construction. A dozen X-ray structures have been deposited to ultra-high resolution (i.e. ≤0.7 Å), for which precise electron density can be exploited to obtain charge density and provide information on the bonding character of catalytic or electron transfer sites. Although the development of neutron macromolecular crystallography over the years has been far less pronounced, and its application much less widespread, the availability of new and improved instrumentation, combined with dedicated deuteration facilities, are beginning to transform the field. Of the 83 macromolecular structures deposited with neutron diffraction data, more than half (49/83, 59%) were released since 2010. Sub-mm3 crystals are now regularly being used for data collection, structures have been determined to atomic resolution for a few small proteins, and much larger unit-cell systems (cell edges >100 Å) are being successfully studied. While some details relating to H-atom positions are tractable with X-ray crystallography at sub-atomic resolution, the mobility of certain H atoms precludes them from being located. In addition, highly polarized H atoms and protons (H+) remain invisible with X-rays. Moreover, the majority of X-ray structures are determined from cryo-cooled crystals at 100 K, and, although radiation damage can be strongly controlled, especially since the advent of shutterless fast detectors, and by using limited doses and crystal translation at micro-focus beams, radiation damage can still take place. Neutron crystallography therefore remains the only approach where diffraction data can be collected at room temperature without radiation damage issues and the only approach to locate mobile or highly polarized H atoms and protons. Here a review of the current status of sub-atomic X-ray and neutron macromolecular crystallography is given and future prospects for combined approaches are outlined. New results from two metalloproteins, copper nitrite reductase and cytochrome c′, are also included, which illustrate the type of information that can be obtained from sub-atomic-resolution (∼0.8 Å) X-ray structures, while also highlighting the need for complementary neutron studies that can provide details of H atoms not provided by X-ray crystallography.
Keywords	neutron ; X-ray ; hydrogen ; proton ; protonation states ; radiation damage ; redox biology ; proton coupling ; electron transfer ; X-ray laser ; XFEL ; Crystallography ; QD901-999
Subject code	500
Language	English
Publishing date	2015-07-01T00:00:00Z
Publisher	International Union of Crystallography
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Covalent narlaprevir- and boceprevir-derived hybrid inhibitors of SARS-CoV-2 main protease

Daniel W. Kneller / Hui Li / Gwyndalyn Phillips / Kevin L. Weiss / Qiu Zhang / Mark A. Arnould / Colleen B. Jonsson / Surekha Surendranathan / Jyothi Parvathareddy / Matthew P. Blakeley / Leighton Coates / John M. Louis / Peter V. Bonnesen / Andrey Kovalevsky

Nature Communications, Vol 13, Iss 1, Pp 1-

2022 Volume 11

Abstract: Three covalent hybrid inhibitors of SARS-CoV-2 main protease (Mpro) have been designed and compared to Pfizer’s nirmatrelvir (PF-07321332), providing atomic and thermodynamic details of their binding to the enzyme, and antiviral potency. ...

Abstract	Three covalent hybrid inhibitors of SARS-CoV-2 main protease (Mpro) have been designed and compared to Pfizer’s nirmatrelvir (PF-07321332), providing atomic and thermodynamic details of their binding to the enzyme, and antiviral potency.
Keywords	Science ; Q
Language	English
Publishing date	2022-04-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: The neutron structure of urate oxidase resolves a long-standing mechanistic conundrum and reveals unexpected changes in protonation.

Esko Oksanen / Matthew P Blakeley / Mohamed El-Hajji / Ulf Ryde / Monika Budayova-Spano

PLoS ONE, Vol 9, Iss 1, p e

2014 Volume 86651

Abstract: Urate oxidase transforms uric acid to 5-hydroxyisourate without the help of cofactors, but the catalytic mechanism has remained enigmatic, as the protonation state of the substrate could not be reliably deduced. We have determined the neutron structure ... ...

Abstract	Urate oxidase transforms uric acid to 5-hydroxyisourate without the help of cofactors, but the catalytic mechanism has remained enigmatic, as the protonation state of the substrate could not be reliably deduced. We have determined the neutron structure of urate oxidase, providing unique information on the proton positions. A neutron crystal structure inhibited by a chloride anion at 2.3 Å resolution shows that the substrate is in fact 8-hydroxyxanthine, the enol tautomer of urate. We have also determined the neutron structure of the complex with the inhibitor 8-azaxanthine at 1.9 Å resolution, showing the protonation states of the K10-T57-H256 catalytic triad. Together with X-ray data and quantum chemical calculations, these structures allow us to identify the site of the initial substrate protonation and elucidate why the enzyme is inhibited by a chloride anion.
Keywords	Medicine ; R ; Science ; Q
Subject code	500
Language	English
Publishing date	2014-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Catalytically important damage-free structures of a copper nitrite reductase obtained by femtosecond X-ray laser and room-temperature neutron crystallography

Thomas P. Halsted / Keitaro Yamashita / Chai C. Gopalasingam / Rajesh T. Shenoy / Kunio Hirata / Hideo Ago / Go Ueno / Matthew P. Blakeley / Robert R. Eady / Svetlana V. Antonyuk / Masaki Yamamoto / S. Samar Hasnain

IUCrJ, Vol 6, Iss 4, Pp 761-

2019 Volume 772

Abstract: Copper-containing nitrite reductases (CuNiRs) that convert NO2− to NO via a CuCAT–His–Cys–CuET proton-coupled redox system are of central importance in nitrogen-based energy metabolism. These metalloenzymes, like all redox enzymes, are very susceptible ... ...

Abstract	Copper-containing nitrite reductases (CuNiRs) that convert NO2− to NO via a CuCAT–His–Cys–CuET proton-coupled redox system are of central importance in nitrogen-based energy metabolism. These metalloenzymes, like all redox enzymes, are very susceptible to radiation damage from the intense synchrotron-radiation X-rays that are used to obtain structures at high resolution. Understanding the chemistry that underpins the enzyme mechanisms in these systems requires resolutions of better than 2 Å. Here, for the first time, the damage-free structure of the resting state of one of the most studied CuNiRs was obtained by combining X-ray free-electron laser (XFEL) and neutron crystallography. This represents the first direct comparison of neutron and XFEL structural data for any protein. In addition, damage-free structures of the reduced and nitrite-bound forms have been obtained to high resolution from cryogenically maintained crystals by XFEL crystallography. It is demonstrated that AspCAT and HisCAT are deprotonated in the resting state of CuNiRs at pH values close to the optimum for activity. A bridging neutral water (D2O) is positioned with one deuteron directed towards AspCAT Oδ1 and one towards HisCAT N∊2. The catalytic T2Cu-ligated water (W1) can clearly be modelled as a neutral D2O molecule as opposed to D3O+ or OD−, which have previously been suggested as possible alternatives. The bridging water restricts the movement of the unprotonated AspCAT and is too distant to form a hydrogen bond to the O atom of the bound nitrite that interacts with AspCAT. Upon the binding of NO2− a proton is transferred from the bridging water to the Oδ2 atom of AspCAT, prompting electron transfer from T1Cu to T2Cu and reducing the catalytic redox centre. This triggers the transfer of a proton from AspCAT to the bound nitrite, enabling the reaction to proceed.
Keywords	copper-containing nitrite reductases ; neutron crystallography ; X-ray free-electron lasers ; Science ; Q
Subject code	500
Language	English
Publishing date	2019-07-01T00:00:00Z
Publisher	International Union of Crystallography
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: A molecular mechanism for transthyretin amyloidogenesis

Ai Woon Yee / Matteo Aldeghi / Matthew P. Blakeley / Andreas Ostermann / Philippe J. Mas / Martine Moulin / Daniele de Sanctis / Matthew W. Bowler / Christoph Mueller-Dieckmann / Edward P. Mitchell / Michael Haertlein / Bert L. de Groot / Elisabetta Boeri Erba / V. Trevor Forsyth

Nature Communications, Vol 10, Iss 1, Pp 1-

2019 Volume 10

Abstract: A number of disease-causing human transthyretin (TTR) mutations are known to lead to amyloid formation. Here the authors combine neutron crystallography, native mass spectrometry and modelling studies to characterize the T119M and S52P-TTR mutants, ... ...

Abstract	A number of disease-causing human transthyretin (TTR) mutations are known to lead to amyloid formation. Here the authors combine neutron crystallography, native mass spectrometry and modelling studies to characterize the T119M and S52P-TTR mutants, providing mechanistic insights into TTR amyloidosis.
Keywords	Science ; Q
Language	English
Publishing date	2019-02-01T00:00:00Z
Publisher	Nature Publishing Group
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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