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Open Access
Abstract: Spike proteins protrude from the SARS-CoV-2 viral envelope and are responsible for initiating fusion into human epithelial cells by binding Angiotensin-Converting Enzyme 2 receptors on the host cell surface. Due to their exposed location on the outside of the virion and their key role in infection, SARS-CoV-2 spike proteins are an important target for vaccine development and drug design. Over the last two years, many spike protein structures have been experimentally determined, providing essential details into the complex structural rearrangements that occur after receptor binding and during fusion of the virion with the host cell, as well as into the interactions of spike protein molecules with antibodies. SARS-CoV-2 variants, particularly those associated with reduced vaccine efficacy, are strongly associated with mutations in two domains of the SARS-CoV-2 spike protein, namely the receptor binding domain and the N-terminal domain, which have both been structurally characterized. This review provides a comprehensive overview of the structural knowledge acquired over the past four years on the SARS-CoV-2 spike protein and its critical role in viral infection.
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Jul 2024
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I04-Macromolecular Crystallography
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George H.
Hutchins
,
Sebastian
Kiehstaller
,
Pascal
Poc
,
Abigail H.
Lewis
,
Jisun
Oh
,
Raya
Sadighi
,
Nicholas M.
Pearce
,
Mohamed
Ibrahim
,
Ivana
Drienovská
,
Anouk M.
Rijs
,
Saskia
Neubacher
,
Sven
Hennig
,
Tom N.
Grossmann
Diamond Proposal Number(s):
[25413]
Open Access
Abstract: Proteins are essential biomolecules and central to biotechnological applications. In many cases, assembly into higher-order structures is a prerequisite for protein function. Under conditions relevant for applications, protein integrity is often challenged, resulting in disassembly, aggregation, and loss of function. The stabilization of quaternary structure has proven challenging, particularly for trimeric and higher-order complexes, given the complexity of involved inter- and intramolecular interaction networks. Here, we describe the chemical bicyclization of homotrimeric protein complexes, thereby increasing protein resistance toward thermal and chemical stress. This approach involves the structure-based selection of cross-linking sites, their variation to cysteine, and a subsequent reaction with a triselectrophilic agent to form a protein assembly with bicyclic topology. Besides overall increased stability, we observe resistance toward aggregation and greatly prolonged shelf life. This bicyclization strategy gives rise to unprecedented protein chain topologies and can enable new biotechnological and biomedical applications.
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Nov 2023
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Thomas M.
Wood
,
Matthieu R.
Zeronian
,
Ned
Buijs
,
Kristine
Bertheussen
,
Hanieh K.
Abedian
,
Aidan V.
Johnson
,
Nicholas M.
Pearce
,
Martin
Lutz
,
Johan
Kemmink
,
Tjalling
Seirsma
,
Leendert W.
Hamoen
,
Bert J. C.
Janssen
,
Nathaniel I.
Martin
Diamond Proposal Number(s):
[19800]
Open Access
Abstract: The continued rise of antibiotic resistance is a global concern that threatens to undermine many aspects of modern medical practice. Key to addressing this threat is the discovery and development of new antibiotics that operate by unexploited modes of action. The so-called calcium-dependent lipopeptide antibiotics (CDAs) are an important emerging class of natural products that provides a source of new antibiotic agents rich in structural and mechanistic diversity. Notable in this regard is the subset of CDAs comprising the laspartomycins and amphomycins/friulimicins that specifically target the bacterial cell wall precursor undecaprenyl phosphate (C55-P). In this study we describe the design and synthesis of new C55-P-targeting CDAs with structural features drawn from both the laspartomycin and amphomycin/friulimicin classes. Assessment of these lipopeptides revealed previously unknown and surprisingly subtle structural features that are required for antibacterial activity. High-resolution crystal structures further indicate that the amphomycin/friulimicin-like lipopeptides adopt a unique crystal packing that governs their interaction with C55-P and provides an explanation for their antibacterial effect. In addition, live-cell microscopy studies provide further insights into the biological activity of the C55-P targeting CDAs highlighting their unique mechanism of action relative to the clinically used CDA daptomycin.
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Feb 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Muhamamd
Faheem
,
Napoleao
Fonseca Valadares
,
Jose
Brandao-Neto
,
Domenico
Bellini
,
Patrick
Collins
,
Nicholas M.
Pearce
,
Louise
Bird
,
Juliana
Torini De Souza
,
Raymond
Owens
,
Humberto
Pereira
,
Frank
Von Delft
,
João Alexandre Ribeiro Gonçalves
Barbosa
Diamond Proposal Number(s):
[11175]
Open Access
Abstract: Several Schistosoma species cause Schistosomiasis, an endemic disease in 78 countries that is ranked second amongst the parasitic diseases in terms of its socioeconomic impact and human health importance. The drug recommended for treatment by the WHO is praziquantel (PZQ), but there are concerns associated with PZQ, such as the lack of information about its exact mechanism of action, its high price, its effectiveness – which is limited to the parasite’s adult form – and reports of resistance. The parasites lack the de novo purine pathway, rendering them dependent on the purine salvage pathway or host purine bases for nucleotide synthesis. Thus, the Schistosoma purine salvage pathway is an attractive target for the development of necessary and selective new drugs. In this study, the purine nucleotide phosphorylase II (PNP2), a new isoform of PNP1, was submitted to a high-throughput fragment-based hit discovery using a crystallographic screening strategy. PNP2 was crystallized and crystals were soaked with 827 fragments, a subset of the Maybridge 1000 library. X-ray diffraction data was collected and structures were solved. Out of 827-screened fragments we have obtained a total of 19 fragments that show binding to PNP2. 14 of these fragments bind to the active site of PNP2, while five were observed in three other sites. Here we present the first fragment screening against PNP2.
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Sep 2021
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NONE-No attached Diamond beamline
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Alice
Douangamath
,
Alisa
Powell
,
Daren
Fearon
,
Patrick M.
Collins
,
Romain
Talon
,
Tobias
Krojer
,
Rachael
Skyner
,
Jose
Brandao-Neto
,
Louise
Dunnett
,
Alexandre
Dias
,
Anthony
Aimon
,
Nicholas M.
Pearce
,
Conor
Wild
,
Tyler J.
Gorrie-Stone
,
Frank
Von Delft
Open Access
Abstract: In fragment-based drug discovery, hundreds or often thousands of compounds smaller than ~300 Da are tested against the protein of interest to identify chemical entities that can be developed into potent drug candidates. Since the compounds are small, interactions are weak, and the screening method must therefore be highly sensitive; moreover, structural information tends to be crucial for elaborating these hits into lead-like compounds. Therefore, protein crystallography has always been a gold-standard technique, yet historically too challenging to find widespread use as a primary screen.
Initial XChem experiments were demonstrated in 2014 and then trialed with academic and industrial collaborators to validate the process. Since then, a large research effort and significant beamtime have streamlined sample preparation, developed a fragment library with rapid follow-up possibilities, automated and improved the capability of I04-1 beamline for unattended data collection, and implemented new tools for data management, analysis and hit identification.
XChem is now a facility for large-scale crystallographic fragment screening, supporting the entire crystals-to-deposition process, and accessible to academic and industrial users worldwide. The peer-reviewed academic user program has been actively developed since 2016, to accommodate projects from as broad a scientific scope as possible, including well-validated as well as exploratory projects. Academic access is allocated through biannual calls for peer-reviewed proposals, and proprietary work is arranged by Diamond's Industrial Liaison group. This workflow has already been routinely applied to over a hundred targets from diverse therapeutic areas, and effectively identifies weak binders (1%-30% hit rate), which both serve as high-quality starting points for compound design and provide extensive structural information on binding sites. The resilience of the process was demonstrated by continued screening of SARS-CoV-2 targets during the COVID-19 pandemic, including a 3-week turn-around for the main protease.
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May 2021
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I04-Macromolecular Crystallography
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Mathias
Wendt
,
Rosa
Bellavita
,
Alan
Gerber
,
Nina-Louisa
Efrém
,
Thirza
Van Ramshorst
,
Nicholas M.
Pearce
,
Paul R. J.
Davey
,
Isabel
Everard
,
Mercedes
Vazquez-Chantada
,
Elisabetta
Chiarpari
,
Paolo
Grieco
,
Sven
Hennig
,
Tom N.
Grossmann
Diamond Proposal Number(s):
[19800]
Open Access
Abstract: Protein complexes are defined by the three‐dimensional structure of participating binding partners. Knowledge about these structures can facilitate the design of peptidomimetics which have been applied e.g. as inhibitors of protein‐protein interactions (PPIs). Even though β‐sheets participate widely in PPIs, they have only rarely served as the basis for peptidomimetic PPI inhibitors, in particular when addressing intracellular targets. Here, we present the structure‐based design of β‐sheet mimetics targeting the intracellular protein β‐catenin, a central component of the Wnt signaling pathway. Based on a protein binding partner of β‐catenin, a macrocyclic peptide was designed and its crystal structure in complex with β‐catenin obtained. Using this structure, we designed a library of bicyclic β‐sheet mimetics employing a late‐stage diversification strategy. Several mimetics were identified that compete with transcription factor binding to β‐catenin and inhibit Wnt signaling in cells. The presented design strategy can support the development of inhibitors for other β‐sheet‐mediated PPIs.
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Mar 2021
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Wout
Oosterheert
,
Katerina T.
Xenaki
,
Viviana
Neviani
,
Wouter
Pos
,
Sofia
Doulkeridou
,
Jip
Manshande
,
Nicholas M.
Pearce
,
Loes M. J.
Kroon-Batenburg
,
Martin
Lutz
,
Paul M. P.
Van Bergen En Henegouwen
,
Piet
Gros
Diamond Proposal Number(s):
[19800]
Open Access
Abstract: Tetraspanins are eukaryotic membrane proteins that contribute to a variety of signaling processes by organizing partner-receptor molecules in the plasma membrane. How tetraspanins bind and cluster partner receptors into tetraspanin-enriched microdomains is unknown. Here, we present crystal structures of the large extracellular loop of CD9 bound to nanobodies 4C8 and 4E8 and, the cryo-EM structure of 4C8-bound CD9 in complex with its partner EWI-F. CD9–EWI-F displays a tetrameric arrangement with two central EWI-F molecules, dimerized through their ectodomains, and two CD9 molecules, one bound to each EWI-F transmembrane helix through CD9-helices h3 and h4. In the crystal structures, nanobodies 4C8 and 4E8 bind CD9 at loops C and D, which is in agreement with the 4C8 conformation in the CD9–EWI-F complex. The complex varies from nearly twofold symmetric (with the two CD9 copies nearly anti-parallel) to ca. 50° bent arrangements. This flexible arrangement of CD9–EWI-F with potential CD9 homo-dimerization at either end provides a “concatenation model” for forming short linear or circular assemblies, which may explain the occurrence of tetraspanin-enriched microdomains.
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Sep 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[15751]
Open Access
Abstract: Allostery is an inherent feature of proteins, but it remains challenging to reveal the mechanisms by which allosteric signals propagate. A clearer understanding of this intrinsic circuitry would afford new opportunities to modulate protein function. Here we have identified allosteric sites in protein tyrosine phosphatase 1B (PTP1B) by combining multiple-temperature X-ray crystallography experiments and structure determination from hundreds of individual small-molecule fragment soaks. New modeling approaches reveal 'hidden' low-occupancy conformational states for protein and ligands. Our results converge on allosteric sites that are conformationally coupled to the active-site WPD loop and are hotspots for fragment binding. Targeting one of these sites with covalently tethered molecules or mutations allosterically inhibits enzyme activity. Overall, this work demonstrates how the ensemble nature of macromolecular structure, revealed here by multitemperature crystallography, can elucidate allosteric mechanisms and open new doors for long-range control of protein function.
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Jun 2018
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I19-Small Molecule Single Crystal Diffraction
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Diamond Proposal Number(s):
[8618]
Abstract: The structural characterization of single crystals of di-4-pyridyl-substituted 3,4,9,10-perylenetetracarboxylic diimide reveals a surprising triple helical arrangement. The intermolecular interactions that lead to such an arrangement are investigated by Hirshfeld surface analysis and indicate that the supramolecular structure arises due to a combination of C–H···O interactions and π–π stacking interactions between adjacent perylene diimide (PDI) species. The interplay of these interactions leads to the formation of a tubular structure enclosed by the triple helix of PDI molecules. In contrast, the analogous phenyl-substituted molecule forms a simple one-dimensional stack of PDI molecules which is also unusual in that the perylene core adopts an essentially planar arrangement despite bay substitution.
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Jan 2018
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[8421]
Open Access
Abstract: Crystallographic fragment screening uses low molecular weight compounds to probe the protein surface and although individual protein-fragment interactions are high quality, fragments commonly bind at low occupancy, historically making identification difficult. However, our new Pan-Dataset Density Analysis method readily identifies binders missed by conventional analysis: for fragment screening data of lysine-specific demethylase 4D (KDM4D), the hit rate increased from 0.9% to 10.6%. Previously unidentified fragments reveal multiple binding sites and demonstrate: the versatility of crystallographic fragment screening; that surprisingly large conformational changes are possible in crystals; and that low crystallographic occupancy does not by itself reflect a protein-ligand complex's significance.
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May 2017
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