I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Martina
Wirth
,
Stephane
Mouilleron
,
Wenxin
Zhang
,
Eva
Sjøttem
,
Yakubu
Princely Abudu
,
Ashish
Jain
,
Hallvard
Lauritz Olsvik
,
Jack-Ansgar
Bruun
,
Minoo
Razi
,
Harold B. J.
Jefferies
,
Rebecca
Lee
,
Dhira
Joshi
,
Nicola
O'Reilly
,
Terje
Johansen
,
Sharon A.
Tooze
Diamond Proposal Number(s):
[9826]
Open Access
Abstract: Autophagy is a highly conserved degradative pathway, essential for cellular homeostasis and implicated in diseases including cancer and neurodegeneration. Autophagy-related 8 (ATG8) proteins play a central role in autophagosome formation and selective delivery of cytoplasmic cargo to lysosomes by recruiting autophagy adaptors and receptors. The LC3-interacting region (LIR) docking site (LDS) of ATG8 proteins binds to LIR motifs present in autophagy adaptors and receptors. LIR-ATG8 interactions can be highly selective for specific mammalian ATG8 family members (LC3A-C, GABARAP, and GABARAPL1-2) and how this specificity is generated and regulated is incompletely understood.
We have identified a LIR motif in the Golgi protein SCOC (short coiled-coil protein) exhibiting strong binding to GABARAP, GABARAPL1, LC3A and LC3C. The residues within and surrounding the core LIR motif of the SCOC LIR domain were phosphorylated by autophagy-related kinases (ULK1-3, TBK1) increasing specifically LC3 family binding. More distant flanking residues also contributed to ATG8 binding. Loss of these residues was compensated by phosphorylation of serine residues immediately adjacent to the core LIR motif, indicating that the interactions of the flanking LIR regions with the LDS are important and highly dynamic.
Our comprehensive structural, biophysical and biochemical analyses support and provide novel mechanistic insights into how phosphorylation of LIR domain residues regulates the affinity and binding specificity of ATG8 proteins towards autophagy adaptors and receptors.
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Jun 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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Diamond Proposal Number(s):
[12342, 23269]
Open Access
Abstract: The rational design of linear peptides that assemble controllably and predictably in water is challenging. Short sequences must encode unique target structures and avoid alternative states. However, the non-covalent forces that stabilize and discriminate between states are weak. Nonetheless, for α-helical coiled-coil assemblies considerable progress has been made in rational de novo design. In these, sequence repeats of nominally hydrophobic (h) and polar (p) residues, hpphppp, direct the assembly of amphipathic helices into dimeric to tetrameric bundles. Expanding this pattern to hpphhph can produce larger α-helical barrels. Here, we show that pentameric to nonameric barrels are accessed by varying the residue at one of the h sites. In peptides with four L/I–K–E–I–A–x–Z repeats, decreasing the size of Z from threonine to serine to alanine to glycine gives progressively larger oligomers. X-ray crystal structures of the resulting α-helical barrels rationalize this: side chains at Z point directly into the helical interfaces, and smaller residues allow closer helix contacts and larger assemblies.
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Apr 2021
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[12342]
Abstract: Rational protein design requires understanding the contribution of each amino acid to a targeted protein fold. For a subset of protein structures, namely, α-helical coiled coils (CCs), knowledge is sufficiently advanced to allow the rational de novo design of many structures, including entirely new protein folds. Current CC design rules center on using aliphatic hydrophobic residues predominantly to drive the folding and assembly of amphipathic α helices. The consequences of using aromatic residues—which would be useful for introducing structural probes, and binding and catalytic functionalities—into these interfaces are not understood. There are specific examples of designed CCs containing such aromatic residues, e.g., phenylalanine-rich sequences, and the use of polar aromatic residues to make buried hydrogen-bond networks. However, it is not known generally if sequences rich in tyrosine can form CCs, or what CC assemblies these would lead to. Here, we explore tyrosine-rich sequences in a general CC-forming background and resolve new CC structures. In one of these, an antiparallel tetramer, the tyrosine residues are solvent accessible and pack at the interface between the core and the surface. In another more complex structure, the residues are buried and form an extended hydrogen-bond network.
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Apr 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[18548]
Open Access
Abstract: Viral diseases pose major threats to humans and other animals, including the billions of chickens that are an important food source as well as a public health concern due to zoonotic pathogens. Unlike humans and other typical mammals, the major histocompatibility complex (MHC) of chickens can confer decisive resistance or susceptibility to many viral diseases. An iconic example is Marek’s disease, caused by an oncogenic herpesvirus with over 100 genes. Classical MHC class I and class II molecules present antigenic peptides to T lymphocytes, and it has been hard to understand how such MHC molecules could be involved in susceptibility to Marek’s disease, given the potential number of peptides from over 100 genes. We used a new in vitro infection system and immunopeptidomics to determine peptide motifs for the 2 class II molecules expressed by the MHC haplotype B2, which is known to confer resistance to Marek’s disease. Surprisingly, we found that the vast majority of viral peptide epitopes presented by chicken class II molecules arise from only 4 viral genes, nearly all having the peptide motif for BL2*02, the dominantly expressed class II molecule in chickens. We expressed BL2*02 linked to several Marek’s disease virus (MDV) peptides and determined one X-ray crystal structure, showing how a single small amino acid in the binding site causes a crinkle in the peptide, leading to a core binding peptide of 10 amino acids, compared to the 9 amino acids in all other reported class II molecules. The limited number of potential T cell epitopes from such a complex virus can explain the differential MHC-determined resistance to MDV, but raises questions of mechanism and opportunities for vaccine targets in this important food species, as well as providing a basis for understanding class II molecules in other species including humans.
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Apr 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Marion
Schuller
,
Galen J.
Correy
,
Stefan
Gahbauer
,
Daren
Fearon
,
Taiasean
Wu
,
Roberto Efraín
Díaz
,
Iris D.
Young
,
Luan
Carvalho Martins
,
Dominique H.
Smith
,
Ursula
Schulze-Gahmen
,
Tristan W.
Owens
,
Ishan
Deshpande
,
Gregory E.
Merz
,
Aye C.
Thwin
,
Justin T.
Biel
,
Jessica K.
Peters
,
Michelle
Moritz
,
Nadia
Herrera
,
Huong T.
Kratochvil
,
Anthony
Aimon
,
James
Bennett
,
Jose
Brandao Neto
,
Aina E.
Cohen
,
Alexandre
Dias
,
Alice
Douangamath
,
Louise
Dunnett
,
Oleg
Fedorov
,
Matteo P.
Ferla
,
Martin R.
Fuchs
,
Tyler J.
Gorrie-Stone
,
James M.
Holton
,
Michael G.
Johnson
,
Tobias
Krojer
,
George
Meigs
,
Alisa J.
Powell
,
Johannes Gregor Matthias
Rack
,
Victor
Rangel
,
Silvia
Russi
,
Rachael E.
Skyner
,
Clyde A.
Smith
,
Alexei S.
Soares
,
Jennifer L.
Wierman
,
Kang
Zhu
,
Peter
O’brien
,
Natalia
Jura
,
Alan
Ashworth
,
John J.
Irwin
,
Michael C.
Thompson
,
Jason E.
Gestwicki
,
Frank
Von Delft
,
Brian K.
Shoichet
,
James S.
Fraser
,
Ivan
Ahel
Diamond Proposal Number(s):
[27001]
Open Access
Abstract: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) macrodomain within the nonstructural protein 3 counteracts host-mediated antiviral adenosine diphosphate–ribosylation signaling. This enzyme is a promising antiviral target because catalytic mutations render viruses nonpathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of 2533 diverse fragments resulted in 214 unique macrodomain-binders. An additional 60 molecules were selected from docking more than 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several fragment hits were confirmed by solution binding using three biophysical techniques (differential scanning fluorimetry, homogeneous time-resolved fluorescence, and isothermal titration calorimetry). The 234 fragment structures explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.
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Apr 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Open Access
Abstract: We have developed a high‐throughput system to synthesise and explore up to 96 heterogeneous catalysts at the same time. The system was developed as a proof of concept, using a standard glass plate and a 3D printed 96‐well plate. Nano‐droplets of catalyst formulations were transferred to the glass plate using an acoustic liquid handler and upon heat treatments, the miniature mesoporous metal oxide (MMO) catalysts were formed. The 3D printed bottomless 96‐well plate was fixed to the glass plate, to give 96 individual wells, each containing a catalyst. Four catalyst plates were prepared (Co3O4‐, Au/Co3O4‐, Pd/Co3O4‐ and Co/Mn‐MMO) to be screened for their activity in the oxidation of morin, as a model reaction. The observed reaction rates (kobs) for each catalyst were calculated to identify the most active catalyst. The general method described herein requires microscopic amounts of catalysts with derivates of the catalyst's composition.
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Apr 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Claire C.
Munier
,
Leonardo
De Maria
,
Karl
Edman
,
Anders
Gunnarsson
,
Marianna
Longo
,
Carol
Mackintosh
,
Saleha
Patel
,
Arjan
Snijder
,
Lisa
Wissler
,
Luc
Brunsveld
,
Christian
Ottmann
,
Matthew W. D.
Perry
Diamond Proposal Number(s):
[20016]
Open Access
Abstract: Glucocorticoid receptor (GR) is a ligand-dependent transcription factor that plays a central role in inflammation. GR activity is also modulated via protein–protein interactions, including binding of 14-3-3 proteins induced by GR phosphorylation. However, the specific phosphorylation sites on GR that trigger these interactions and their functional consequences are less clear. Hence, we sought to examine this system in more detail. We used phosphorylated GR peptides, biophysical studies and X-ray crystallography to identify key residues within the ligand binding domain of GR, T524 and S617, whose phosphorylation results in binding of the representative 14-3-3 protein 14-3-3ζ. A kinase screen identified MINK1 as responsible for phosphorylating T524 and ROCK1 for phosphorylating S617; cell-based approaches confirmed the importance of both GR phosphosites and MINK1 but not ROCK1 alone in inducing GR–14-3-3 binding. Together our results provide molecular-level insight into 14-3-3-mediated regulation of GR and highlight both MINK1 and the GR–14-3-3 axis as potential targets for future therapeutic intervention.
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Mar 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Derun
Li
,
Yongqi
Deng
,
Abdelghani
Achab
,
Indu
Bharathan
,
Brett Andrew
Hopkins
,
Wensheng
Yu
,
Hongjun
Zhang
,
Sulagna
Sanyal
,
Qinglin
Pu
,
Hua
Zhou
,
Kun
Liu
,
Jongwon
Lim
,
Xavier
Fradera
,
Charles A.
Lesburg
,
Alfred
Lammens
,
Theodore A.
Martinot
,
Ryan D.
Cohen
,
Amy C.
Doty
,
Heidi
Ferguson
,
Elliott B.
Nickbarg
,
Mangeng
Cheng
,
Peter
Spacciapoli
,
Prasanthi
Geda
,
Xuelei
Song
,
Nadya
Smotrov
,
Pravien
Abeywickrema
,
Christine
Andrews
,
Chad
Chamberlin
,
Omar
Mabrouk
,
Patrick
Curran
,
Matthew
Richards
,
Peter
Saradjian
,
J. Richard
Miller
,
Ian
Knemeyer
,
Karin M.
Otte
,
Stella
Vincent
,
Nunzio
Sciammetta
,
Alexander
Pasternak
,
David Jonathan
Bennett
,
Yongxin
Han
Abstract: Indoleamine-2,3-dioxygenase-1 (IDO1) has emerged as an attractive target for cancer immunotherapy. An automated ligand identification system screen afforded the tetrahydroquinoline class of novel IDO1 inhibitors. Potency and pharmacokinetic (PK) were key issues with this class of compounds. Structure-based drug design and strategic incorporation of polarity enabled the rapid improvement on potency, solubility, and oxidative metabolic stability. Metabolite identification studies revealed that amide hydrolysis in the D-pocket was the key clearance mechanism for this class. Strategic survey of amide isosteres revealed that carbamates and N-pyrimidines, which maintained exquisite potencies, mitigated the amide hydrolysis issue and led to an improved rat PK profile. The lead compound 28 is a potent IDO1 inhibitor, with clean off-target profiles and the potential for quaque die dosing in humans.
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Mar 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8492, 11265]
Open Access
Abstract: Microsomal glutathione S-transferase 2 (MGST2) produces leukotriene C4, key for intracrine signaling of endoplasmic reticulum (ER) stress, oxidative DNA damage and cell death. MGST2 trimer restricts catalysis to only one out of three active sites at a time, but the molecular basis is unknown. Here, we present crystal structures of human MGST2 combined with biochemical and computational evidence for a concerted mechanism, involving local unfolding coupled to global conformational changes that regulate catalysis. Furthermore, synchronized changes in the biconical central pore modulate the hydrophobicity and control solvent influx to optimize reaction conditions at the active site. These unique mechanistic insights pertain to other, structurally related, drug targets.
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Mar 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Olga V.
Moroz
,
Elena
Blagova
,
Edward
Taylor
,
Johan
Turkenburg
,
Lars K.
Skov
,
Garry P.
Gippert
,
Kirk M.
Schnorr
,
Li
Ming
,
Liu
Ye
,
Mikkel
Klausen
,
Marianne T.
Cohn
,
Esben G. W.
Schmidt
,
Søren
Nymand-Grarup
,
Gideon J.
Davies
,
Keith S.
Wilson
Diamond Proposal Number(s):
[13587, 7864]
Open Access
Abstract: Muramidases/lysozymes hydrolyse the peptidoglycan component of the bacterial cell wall. They are found in many of the glycoside hydrolase (GH) families. Family GH25 contains muramidases/lysozymes, known as CH type lysozymes, as they were initially discovered in the Chalaropsis species of fungus. The characterized enzymes from GH25 exhibit both β-1,4-N-acetyl- and β-1,4-N,6-O-diacetylmuramidase activities, cleaving the β-1,4-glycosidic bond between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) moieties in the carbohydrate backbone of bacterial peptidoglycan. Here, a set of fungal GH25 muramidases were identified from a sequence search, cloned and expressed and screened for their ability to digest bacterial peptidoglycan, to be used in a commercial application in chicken feed. The screen identified the enzyme from Acremonium alcalophilum JCM 736 as a suitable candidate for this purpose and its relevant biochemical and biophysical and properties are described. We report the crystal structure of the A. alcalophilum enzyme at atomic, 0.78 Å resolution, together with that of its homologue from Trichobolus zukalii at 1.4 Å, and compare these with the structures of homologues. GH25 enzymes offer a new solution in animal feed applications such as for processing bacterial debris in the animal gut.
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Mar 2021
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