I04-Macromolecular Crystallography
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Han Wee
Ong
,
Yi
Liang
,
William
Richardson
,
Emily R.
Lowry
,
Carrow I.
Wells
,
Xiangrong
Chen
,
Margaux
Silvestre
,
Kelvin
Dempster
,
Josie A.
Silvaroli
,
Jeffery L.
Smith
,
Hynek
Wichterle
,
Navjot S.
Pabla
,
Sila K.
Ultanir
,
Alex N.
Bullock
,
David H.
Drewry
,
Alison D.
Axtman
Diamond Proposal Number(s):
[28172]
Open Access
Abstract: Despite mediating several essential processes in the brain, including during development, cyclin-dependent kinase-like 5 (CDKL5) remains a poorly characterized human protein kinase. Accordingly, its substrates, functions, and regulatory mechanisms have not been fully described. We realized that availability of a potent and selective small molecule probe targeting CDKL5 could enable illumination of its roles in normal development as well as in diseases where it has become aberrant due to mutation. We prepared analogs of AT-7519, a compound that has advanced to phase II clinical trials and is a known inhibitor of several cyclin-dependent kinases (CDKs) and cyclin-dependent kinase-like kinases (CDKLs). We identified analog 2 as a highly potent and cell-active chemical probe for CDKL5/GSK3 (glycogen synthase kinase 3). Evaluation of its kinome-wide selectivity confirmed that analog 2 demonstrates excellent selectivity and only retains GSK3α/β affinity. We next demonstrated the inhibition of downstream CDKL5 and GSK3α/β signaling and solved a co-crystal structure of analog 2 bound to human CDKL5. A structurally similar analog (4) proved to lack CDKL5 affinity and maintain potent and selective inhibition of GSK3α/β, making it a suitable negative control. Finally, we used our chemical probe pair (2 and 4) to demonstrate that inhibition of CDKL5 and/or GSK3α/β promotes the survival of human motor neurons exposed to endoplasmic reticulum stress. We have demonstrated a neuroprotective phenotype elicited by our chemical probe pair and exemplified the utility of our compounds to characterize the role of CDKL5/GSK3 in neurons and beyond.
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Apr 2023
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Open Access
Abstract: Synaptic vesicle (SV) fusion with the plasma membrane (PM) proceeds through intermediate steps that remain poorly resolved. The effect of persistent high or low exocytosis activity on intermediate steps remains unknown. Using spray-mixing plunge-freezing cryo-electron tomography we observe events following synaptic stimulation at nanometer resolution in near-native samples. Our data suggest that during the stage that immediately follows stimulation, termed early fusion, PM and SV membrane curvature changes to establish a point contact. The next stage—late fusion—shows fusion pore opening and SV collapse. During early fusion, proximal tethered SVs form additional tethers with the PM and increase the inter-SV connector number. In the late-fusion stage, PM-proximal SVs lose their interconnections, allowing them to move toward the PM. Two SNAP-25 mutations, one arresting and one disinhibiting spontaneous release, cause connector loss. The disinhibiting mutation causes loss of membrane-proximal multiple-tethered SVs. Overall, tether formation and connector dissolution are triggered by stimulation and respond to spontaneous fusion rate manipulation. These morphological observations likely correspond to SV transition from one functional pool to another.
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Mar 2023
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Krios IV-Titan Krios IV at Diamond
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Yang
Yang
,
Holly J.
Garringer
,
Yang
Shi
,
Sofia
Lovestam
,
Sew
Peak-Chew
,
Xianjun
Zhang
,
Abhay
Kotecha
,
Mehtap
Bacioglu
,
Atsuo
Koto
,
Masaki
Takao
,
Maria Grazia
Spillantini
,
Bernardino
Ghetti
,
Ruben
Vidal
,
Alexey G.
Murzin
,
Sjors H. W.
Scheres
,
Michel
Goedert
Diamond Proposal Number(s):
[23268]
Open Access
Abstract: A 21-nucleotide duplication in one allele of SNCA was identified in a previously described disease with abundant α-synuclein inclusions that we now call juvenile-onset synucleinopathy (JOS). This mutation translates into the insertion of MAAAEKT after residue 22 of α-synuclein, resulting in a protein of 147 amino acids. Both wild-type and mutant proteins were present in sarkosyl-insoluble material that was extracted from frontal cortex of the individual with JOS and examined by electron cryo-microscopy. The structures of JOS filaments, comprising either a single protofilament, or a pair of protofilaments, revealed a new α-synuclein fold that differs from the folds of Lewy body diseases and multiple system atrophy (MSA). The JOS fold consists of a compact core, the sequence of which (residues 36–100 of wild-type α-synuclein) is unaffected by the mutation, and two disconnected density islands (A and B) of mixed sequences. There is a non-proteinaceous cofactor bound between the core and island A. The JOS fold resembles the common substructure of MSA Type I and Type II dimeric filaments, with its core segment approximating the C-terminal body of MSA protofilaments B and its islands mimicking the N-terminal arm of MSA protofilaments A. The partial similarity of JOS and MSA folds extends to the locations of their cofactor-binding sites. In vitro assembly of recombinant wild-type α-synuclein, its insertion mutant and their mixture yielded structures that were distinct from those of JOS filaments. Our findings provide insight into a possible mechanism of JOS fibrillation in which mutant α-synuclein of 147 amino acids forms a nucleus with the JOS fold, around which wild-type and mutant proteins assemble during elongation.
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Feb 2023
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Krios I-Titan Krios I at Diamond
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Yang
Yang
,
Wenjuan
Zhang
,
Alexey G.
Murzin
,
Manuel
Schweighauser
,
Melissa
Huang
,
Sofia
Lovestam
,
Sew Y.
Peak-Chew
,
Takashi
Saito
,
Takaomi C.
Saido
,
Jennifer
Macdonald
,
Isabelle
Lavenir
,
Bernardino
Ghetti
,
Caroline
Graff
,
Amit
Kumar
,
Agneta
Nordberg
,
Michel
Goedert
,
Sjors H. W.
Scheres
Diamond Proposal Number(s):
[23268]
Open Access
Abstract: The Arctic mutation, encoding E693G in the amyloid precursor protein (APP) gene [E22G in amyloid-β (Aβ)], causes dominantly inherited Alzheimer’s disease. Here, we report the high-resolution cryo-EM structures of Aβ filaments from the frontal cortex of a previously described case (AβPParc1) with the Arctic mutation. Most filaments consist of two pairs of non-identical protofilaments that comprise residues V12–V40 (human Arctic fold A) and E11–G37 (human Arctic fold B). They have a substructure (residues F20–G37) in common with the folds of type I and type II Aβ42. When compared to the structures of wild-type Aβ42 filaments, there are subtle conformational changes in the human Arctic folds, because of the lack of a side chain at G22, which may strengthen hydrogen bonding between mutant Aβ molecules and promote filament formation. A minority of Aβ42 filaments of type II was also present, as were tau paired helical filaments. In addition, we report the cryo-EM structures of Aβ filaments with the Arctic mutation from mouse knock-in line AppNL−G−F. Most filaments are made of two identical mutant protofilaments that extend from D1 to G37 (AppNL−G−F murine Arctic fold). In a minority of filaments, two dimeric folds pack against each other in an anti-parallel fashion. The AppNL−G−F murine Arctic fold differs from the human Arctic folds, but shares some substructure.
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Jan 2023
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13775]
Open Access
Abstract: Tripartite motif (TRIM) proteins constitute a large family of RING-type E3 ligases that share a conserved domain architecture. TRIM2 and TRIM3 are paralogous class VII TRIM members that are expressed mainly in the brain and regulate different neuronal functions. Here we present a detailed structure-function analysis of TRIM2 and TRIM3, which despite high sequence identity, exhibit markedly different self-association and activity profiles. We show that the isolated RING domain of human TRIM3 is monomeric and inactive, and that this lack of activity is due to a few placental mammal-specific amino acid changes adjacent to the core RING domain that prevent self-association but not E2 recognition. We demonstrate that the activity of human TRIM3 RING can be restored by substitution with the relevant region of human TRIM2 or by hetero-dimerization with human TRIM2, establishing that subtle amino acid changes can profoundly affect TRIM protein activity. Finally, we show that TRIM2 and TRIM3 interact in a cellular context via their filamin and coiled-coil domains, respectively.
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Dec 2022
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I24-Microfocus Macromolecular Crystallography
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David A.
Candito
,
Vladimir
Simov
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Anmol
Gulati
,
Solomon
Kattar
,
Ryan W.
Chau
,
Blair T.
Lapointe
,
Joey L.
Methot
,
Duane E.
Demong
,
Thomas H.
Graham
,
Ravi
Kurukulasuriya
,
Mitchell H.
Keylor
,
Ling
Tong
,
Gregori J.
Morriello
,
John J.
Acton
,
Barbara
Pio
,
Weiguo
Liu
,
Jack D.
Scott
,
Michael J.
Ardolino
,
Theodore A.
Martinot
,
Matthew L.
Maddess
,
Xin
Yan
,
Hakan
Gunaydin
,
Rachel L.
Palte
,
Spencer E.
Mcminn
,
Lisa
Nogle
,
Hongshi
Yu
,
Ellen C.
Minnihan
,
Charles A.
Lesburg
,
Ping
Liu
,
Jing
Su
,
Laxminarayan G.
Hegde
,
Lily Y.
Moy
,
Janice D.
Woodhouse
,
Robert
Faltus
,
Tina
Xiong
,
Paul
Ciaccio
,
Jennifer A.
Piesvaux
,
Karin M.
Otte
,
Matthew E.
Kennedy
,
David J.
Bennett
,
Erin F.
Dimauro
,
Matthew J.
Fell
,
Santhosh
Neelamkavil
,
Harold B.
Wood
,
Peter H.
Fuller
,
J. Michael
Ellis
Abstract: Inhibition of leucine-rich repeat kinase 2 (LRRK2) kinase activity represents a genetically supported, chemically tractable, and potentially disease-modifying mechanism to treat Parkinson’s disease. Herein, we describe the optimization of a novel series of potent, selective, central nervous system (CNS)-penetrant 1-heteroaryl-1H-indazole type I (ATP competitive) LRRK2 inhibitors. Type I ATP-competitive kinase physicochemical properties were integrated with CNS drug-like properties through a combination of structure-based drug design and parallel medicinal chemistry enabled by sp3–sp2 cross-coupling technologies. This resulted in the discovery of a unique sp3-rich spirocarbonitrile motif that imparted extraordinary potency, pharmacokinetics, and favorable CNS drug-like properties. The lead compound, 25, demonstrated exceptional on-target potency in human peripheral blood mononuclear cells, excellent off-target kinase selectivity, and good brain exposure in rat, culminating in a low projected human dose and a pre-clinical safety profile that warranted advancement toward pre-clinical candidate enabling studies.
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Dec 2022
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[19800]
Open Access
Abstract: Cell-surface expressed contactin 1 and neurofascin 155 control wiring of the nervous system and interact across cells to form and maintain paranodal myelin-axon junctions. The molecular mechanism of contactin 1 – neurofascin 155 adhesion complex formation is unresolved. Crystallographic structures of complexed and individual contactin 1 and neurofascin 155 binding regions presented here, provide a rich picture of how competing and complementary interfaces, post-translational glycosylation, splice differences and structural plasticity enable formation of diverse adhesion sites. Structural, biophysical, and cell-clustering analysis reveal how conserved Ig1-2 interfaces form competing heterophilic contactin 1 – neurofascin 155 and homophilic neurofascin 155 complexes whereas contactin 1 forms low-affinity clusters through interfaces on Ig3-6. The structures explain how the heterophilic Ig1-Ig4 horseshoe’s in the contactin 1 – neurofascin 155 complex define the 7.4 nm paranodal spacing and how the remaining six domains enable bridging of distinct intercellular distances.
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Nov 2022
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I13-2-Diamond Manchester Imaging
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Francesca
Palermo
,
Nicola
Pieroni
,
Alessia
Sanna
,
Benedetta
Parodi
,
Consuelo
Venturi
,
Ginevra
Begani Provinciali
,
Lorenzo
Massimi
,
Laura
Maugeri
,
Gian Paolo
Marra
,
Elena
Longo
,
Lorenzo
D’amico
,
Giulia
Saccomano
,
Jonathan
Perrin
,
Giuliana
Tromba
,
Inna
Bukreeva
,
Michela
Fratini
,
Giuseppe
Gigli
,
Nicole
Kerlero De Rosbo
,
Alessia
Cedola
Diamond Proposal Number(s):
[23997]
Open Access
Abstract: The 3D complexity of biological tissues and intricate structural-functional connections call for state-of-the-art X-ray imaging approaches to overcome limitations of classical imaging. Unlike other imaging techniques, X-ray phase-contrast tomography (XPCT) offers a highly sensitive 3D imaging approach to investigate different disease-relevant networks at levels ranging from single cell through to intact organ. We present here a concomitant study of the evolution of tissue damage and inflammation in different organs affected by the disease in the murine model for multiple sclerosis, a demyelinating autoimmune disorder of the central nervous system. XPCT identifies and monitors structural and cellular alterations throughout the central nervous system, but also in the gut, and eye, of mice induced to develop multiple sclerosis-like disease and sacrificed at pre-symptomatic and symptomatic time points. This study details the sequential evolution of multi-organ damages in the murine multiple sclerosis model showing the disease development and progression which is of relevance for the human case.
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Nov 2022
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: Channels and transporters are essential proteins for the control, mediation and termination of neurotransmission. These are implicated in numerous pathological conditions, including epilepsy, Parkinson’s disease, neuropathic pain and nicotine addiction. However, the structural and ligand binding aspects of many of these channel and transporter proteins are poorly defined, which limits being able to design new molecules that can effectively target these conditions.
It was aimed to investigate structure and ligand binding at neurotransmitter channels and transporters. The first aims involved elucidating the binding modes and structure-activity relationships of novel ligands at nicotinic acetylcholine receptors (nAChRs), using the surrogate protein acetylcholine binding protein (AChBP). The ligands being characterised were of interest as potential anti-smoking agents and as research tools for studying nAChRs. Binding data and protein complex crystal structures were obtained for several of these novel ligands and it was possible to identify residues which were potentially responsible for their modes of action and affinity to AChBP, and henceforth likely to nAChRs. Knowledge of these interactions could assist in the future design of other ligands targeting nAChRs.
The second set of aims were associated with attempting to establish methodologies for the efficient recombinant production of complex eukaryotic ion channels and neurotransmitter sodium symporters. The initial objective was the insect ligand gated ion channel resistance to dieldrin (RDL), which is a target for insecticides. Sf9 insect cells proved unsuitable for production as only a small amount of the total protein could be extracted with non-ionic detergents and it was implied that the majority of the protein was likely in an un-folded state. Mammalian HEK293 cells were more successful as the protein could be efficiently solubilised, but ultimately the yields of purified protein were too low for this to be a feasible approach.
There was more success with producing the human GABA transporter 1 (GAT1). This terminates the actions of the inhibitory neurotransmitter GABA by removing it from the synapse and is a therapeutic target for the control of epilepsy. Using Sf9 cells and a conventional baculovirus system showed initial success, but there were ultimately problems with aggregation. Use of a recently described baculovirus system with an early Drosophila Hsp70 promoter however resolved these problems and led to high yields of purified GAT1. The obtained protein was suggested to be potentially suitable for future structural studies by single particle cryogenic electron microscopy (cryo-EM). Purified GAT1 was also used as a target to isolate recombinant nanobodies from a yeast library and these may be of assistance for increasing the size of the protein for cryo-EM studies.
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Oct 2022
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Krios IV-Titan Krios IV at Diamond
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Caroline
Neumann
,
Lena
Lindtoft Rosenbæk
,
Rasmus
Kock Flygaard
,
Michael
Habeck
,
Jesper Lykkegaard
Karlsen
,
Yong
Wang
,
Kresten
Lindorff-Larsen
,
Hans Henrik
Gad
,
Rune
Hartmann
,
Joseph A.
Lyons
,
Robert A.
Fenton
,
Poul
Nissen
Diamond Proposal Number(s):
[21404]
Open Access
Abstract: The sodium–potassium–chloride transporter NKCC1 of the SLC12 family performs Na+-dependent Cl−- and K+-ion uptake across plasma membranes. NKCC1 is important for regulating cell volume, hearing, blood pressure, and regulation of hyperpolarizing GABAergic and glycinergic signaling in the central nervous system. Here, we present a 2.6 Å resolution cryo-electron microscopy structure of human NKCC1 in the substrate-loaded (Na+, K+, and 2 Cl−) and occluded, inward-facing state that has also been observed for the SLC6-type transporters MhsT and LeuT. Cl− binding at the Cl1 site together with the nearby K+ ion provides a crucial bridge between the LeuT-fold scaffold and bundle domains. Cl−-ion binding at the Cl2 site seems to undertake a structural role similar to conserved glutamate of SLC6 transporters and may allow for Cl−-sensitive regulation of transport. Supported by functional studies in mammalian cells and computational simulations, we describe a putative Na+ release pathway along transmembrane helix 5 coupled to the Cl2 site. The results provide insight into the structure–function relationship of NKCC1 with broader implications for other SLC12 family members.
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Oct 2022
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