Krios II-Titan Krios II at Diamond
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Huanyu Z.
Li
,
Ashley C. W.
Pike
,
Irina
Lotsaris
,
Gamma
Chi
,
Jesper S.
Hansen
,
Sarah C.
Lee
,
Karin E. J.
Roedstroem
,
Simon R.
Bushell
,
David
Speedman
,
Adam
Evans
,
Dong
Wang
,
Didi
He
,
Leela
Shrestha
,
Chady
Nasrallah
,
Nicola A.
Burgess-Brown
,
Robert J.
Vandenberg
,
Timothy R.
Dafforn
,
Elisabeth P.
Carpenter
,
David B.
Sauer
Diamond Proposal Number(s):
[28713]
Open Access
Abstract: Proline is widely known as the only proteogenic amino acid with a secondary amine. In addition to its crucial role in protein structure, the secondary amino acid modulates neurotransmission and regulates the kinetics of signaling proteins. To understand the structural basis of proline import, we solved the structure of the proline transporter SIT1 in complex with the COVID-19 viral receptor ACE2 by cryo-electron microscopy. The structure of pipecolate-bound SIT1 reveals the specific sequence requirements for proline transport in the SLC6 family and how this protein excludes amino acids with extended side chains. By comparing apo and substrate-bound SIT1 states, we also identify the structural changes that link substrate release and opening of the cytoplasmic gate and provide an explanation for how a missense mutation in the transporter causes iminoglycinuria.
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Jun 2024
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I24-Microfocus Macromolecular Crystallography
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Karin E. J.
Roedstroem
,
Alexander
Cloake
,
Janina
Sörmann
,
Agnese
Baronina
,
Kathryn H. M.
Smith
,
Ashley C. W.
Pike
,
Jackie
Ang
,
Peter
Proks
,
Marcus
Schewe
,
Ingelise
Holland-Kaye
,
Simon R.
Bushell
,
Jenna
Elliott
,
Els
Pardon
,
Thomas
Baukrowitz
,
Raymond J.
Owens
,
Simon
Newstead
,
Jan
Steyaert
,
Elisabeth P.
Carpenter
,
Stephen J.
Tucker
Diamond Proposal Number(s):
[15433, 19301]
Open Access
Abstract: Potassium channels of the Two-Pore Domain (K2P) subfamily, KCNK1-KCNK18, play crucial roles in controlling the electrical activity of many different cell types and represent attractive therapeutic targets. However, the identification of highly selective small molecule drugs against these channels has been challenging due to the high degree of structural and functional conservation that exists not only between K2P channels, but across the whole K+ channel superfamily. To address the issue of selectivity, here we generate camelid antibody fragments (nanobodies) against the TREK-2 (KCNK10) K2P K+ channel and identify selective binders including several that directly modulate channel activity. X-ray crystallography and CryoEM data of these nanobodies in complex with TREK-2 also reveal insights into their mechanisms of activation and inhibition via binding to the extracellular loops and Cap domain, as well as their suitability for immunodetection. These structures facilitate design of a biparatropic inhibitory nanobody with markedly improved sensitivity. Together, these results provide important insights into TREK channel gating and provide an alternative, more selective approach to modulation of K2P channel activity via their extracellular domains.
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May 2024
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Krios IV-Titan Krios IV at Diamond
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Gamma
Chi
,
Dawid
Jaślan
,
Veronika
Kudrina
,
Julia
Böck
,
Huanyu
Li
,
Ashley C. W.
Pike
,
Susanne
Rautenberg
,
Einar
Krogsaeter
,
Tina
Bohstedt
,
Dong
Wang
,
Gavin
Mckinley
,
Alejandra
Fernandez-Cid
,
Shubhashish M. M.
Mukhopadhyay
,
Nicola A.
Burgess-Brown
,
Marco
Keller
,
Franz
Bracher
,
Christian
Grimm
,
Katharina L.
Dürr
Diamond Proposal Number(s):
[20223]
Open Access
Abstract: Two pore channels are lysosomal cation channels with crucial roles in tumor angiogenesis and viral release from endosomes. Inhibition of the two-pore channel 2 (TPC2) has emerged as potential therapeutic strategy for the treatment of cancers and viral infections, including Ebola and COVID-19. Here, we demonstrate that antagonist SG-094, a synthetic analog of the Chinese alkaloid medicine tetrandrine with increased potency and reduced toxicity, induces asymmetrical structural changes leading to a single binding pocket at only one intersubunit interface within the asymmetrical dimer. Supported by functional characterization of mutants by Ca2+ imaging and patch clamp experiments, we identify key residues in S1 and S4 involved in compound binding to the voltage sensing domain II. SG-094 arrests IIS4 in a downward shifted state which prevents pore opening via the IIS4/S5 linker, hence resembling gating modifiers of canonical VGICs. These findings may guide the rational development of new therapeutics antagonizing TPC2 activity.
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May 2024
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I24-Microfocus Macromolecular Crystallography
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Gamma
Chi
,
Larissa
Dietz
,
Haiping
Tang
,
Matthew
Snee
,
Andreea
Scacioc
,
Dong
Wang
,
Gavin
Mckinley
,
Shubhashish M. m.
Mukhopadhyay
,
Ashley C. W.
Pike
,
Rod
Chalk
,
Nicola A.
Burgess-Brown
,
Jean-Pierre
Timmermans
,
Wouter
Van Putte
,
Carol V.
Robinson
,
Katharina L.
Duerr
Open Access
Abstract: In this study, we present the structures of human urea transporters UT-A and UT-B to characterize them at molecular level and to detail the mechanism of UT-B inhibition by its selective inhibitor, UTBinh-14. High-resolution structures of both transporters establish the structural basis for the inhibitor’s selectivity to UT-B, and the identification of multiple binding sites for the inhibitor will aid with the development of drug lead molecules targeting both transporters. Our study also discovers phospholipids associating with the urea transporters by combining structural observations, native MS, and lipidomics analysis. These insights improve our understanding of urea transporter function at a molecular level and provide a blueprint for a structure-guided design of therapeutics targeting these transporters.
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Sep 2023
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I24-Microfocus Macromolecular Crystallography
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Laiyin
Nie
,
Tomas C.
Pascoa
,
Ashley C. W.
Pike
,
Simon R.
Bushell
,
Andrew
Quigley
,
Gian Filippo
Ruda
,
Amy
Chu
,
Victoria
Cole
,
David
Speedman
,
Tiago
Moreira
,
Leela
Shrestha
,
Shubhashish M. M.
Mukhopadhyay
,
Nicola A.
Burgess-Brown
,
James D.
Love
,
Paul E.
Brennan
,
Elisabeth P.
Carpenter
Diamond Proposal Number(s):
[19301]
Abstract: Very long chain fatty acids (VLCFAs) are essential building blocks for the synthesis of ceramides and sphingolipids. The first step in the fatty acid elongation cycle is catalyzed by the 3-keto acyl-coenzyme A (CoA) synthases (in mammals, ELOVL elongases). Although ELOVLs are implicated in common diseases, including insulin resistance, hepatic steatosis and Parkinson’s, their underlying molecular mechanisms are unknown. Here we report the structure of the human ELOVL7 elongase, which comprises an inverted transmembrane barrel surrounding a 35-Å long tunnel containing a covalently attached product analogue. The structure reveals the substrate-binding sites in the narrow tunnel and an active site deep in the membrane. We demonstrate that chain elongation proceeds via an acyl-enzyme intermediate involving the second histidine in the canonical HxxHH motif. The unusual substrate-binding arrangement and chemistry suggest mechanisms for selective ELOVL inhibition, relevant for diseases where VLCFAs accumulate, such as X-linked adrenoleukodystrophy.
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Jun 2021
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I24-Microfocus Macromolecular Crystallography
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Karin E. J.
Roedstroem
,
Aytuğ K.
Kiper
,
Wei
Zhang
,
Susanne
Rinné
,
Ashley C. W.
Pike
,
Matthias
Goldstein
,
Linus J.
Conrad
,
Martina
Delbeck
,
Michael G.
Hahn
,
Heinrich
Meier
,
Magdalena
Platzk
,
Andrew
Quigley
,
David
Speedman
,
Leela
Shrestha
,
Shubhashish M. M.
Mukhopadhyay
,
Nicola A.
Burgess-Brown
,
Stephen J.
Tucker
,
Thomas
Müller
,
Niels
Decher
,
Elisabeth P.
Carpenter
Abstract: TWIK-related acid-sensitive potassium (TASK) channels—members of the two pore domain potassium (K2P) channel family—are found in neurons, cardiomyocytes and vascular smooth muscle cells, where they are involved in the regulation of heart rate, pulmonary artery tone, sleep/wake cycles8 and responses to volatile anaesthetics. K2P channels regulate the resting membrane potential, providing background K+ currents controlled by numerous physiological stimuli. Unlike other K2P channels, TASK channels are able to bind inhibitors with high affinity, exceptional selectivity and very slow compound washout rates. As such, these channels are attractive drug targets, and TASK-1 inhibitors are currently in clinical trials for obstructive sleep apnoea and atrial fibrillation. In general, potassium channels have an intramembrane vestibule with a selectivity filter situated above and a gate with four parallel helices located below; however, the K2P channels studied so far all lack a lower gate. Here we present the X-ray crystal structure of TASK-1, and show that it contains a lower gate—which we designate as an ‘X-gate’—created by interaction of the two crossed C-terminal M4 transmembrane helices at the vestibule entrance. This structure is formed by six residues (243VLRFMT248) that are essential for responses to volatile anaesthetics, neurotransmitters and G-protein-coupled receptors. Mutations within the X-gate and the surrounding regions markedly affect both the channel-open probability and the activation of the channel by anaesthetics. Structures of TASK-1 bound to two high-affinity inhibitors show that both compounds bind below the selectivity filter and are trapped in the vestibule by the X-gate, which explains their exceptionally low washout rates. The presence of the X-gate in TASK channels explains many aspects of their physiological and pharmacological behaviour, which will be beneficial for the future development and optimization of TASK modulators for the treatment of heart, lung and sleep disorders.
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Apr 2020
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Krios I-Titan Krios I at Diamond
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Qinrui
Wang
,
Robin A.
Corey
,
George
Hedger
,
Prafulla
Aryal
,
Mariana
Grieben
,
Chady
Nasrallah
,
Agnese
Baronina
,
Ashley C. W.
Pike
,
Jiye
Shi
,
Elisabeth P.
Carpenter
,
Mark S. P.
Sansom
Diamond Proposal Number(s):
[14856]
Open Access
Abstract: Polycystin-2 (PC2) is a transient receptor potential (TRP) channel present in ciliary membranes of the kidney. PC2 shares a transmembrane fold with other TRP channels, in addition to an extracellular domain found in TRPP and TRPML channels. Using molecular dynamics (MD) simulations and cryoelectron microscopy we identify and characterize PIP2 and cholesterol interactions with PC2. PC2 is revealed to have a PIP binding site close to the equivalent vanilloid/lipid binding site in the TRPV1 channel. A 3.0-Å structure reveals a binding site for cholesterol on PC2. Cholesterol interactions with the channel at this site are characterized by MD simulations. The two classes of lipid binding sites are compared with sites observed in other TRPs and in Kv channels. These findings suggest PC2, in common with other ion channels, may be modulated by both PIPs and cholesterol, and position PC2 within an emerging model of the roles of lipids in the regulation and organization of ciliary membranes.
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Dec 2019
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I24-Microfocus Macromolecular Crystallography
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Simon R.
Bushell
,
Ashley C. W.
Pike
,
Maria E.
Falzone
,
Nils J. G.
Rorsman
,
Chau M.
Ta
,
Robin A.
Corey
,
Thomas D.
Newport
,
John C.
Christianson
,
Lara F.
Scofano
,
Chitra
Shintre
,
Annamaria
Tessitore
,
Amy
Chu
,
Qinrui
Wang
,
Leela
Shrestha
,
Shubhashish M. M.
Mukhopadhyay
,
James D.
Love
,
Nicola A.
Burgess-Brown
,
Rebecca
Sitsapesan
,
Phillip J.
Stansfeld
,
Juha T.
Huiskonen
,
Paolo
Tammaro
,
Alessio
Accardi
,
Elisabeth P.
Carpenter
Diamond Proposal Number(s):
[10619, 15433]
Open Access
Abstract: Membranes in cells have defined distributions of lipids in each leaflet, controlled by lipid scramblases and flip/floppases. However, for some intracellular membranes such as the endoplasmic reticulum (ER) the scramblases have not been identified. Members of the TMEM16 family have either lipid scramblase or chloride channel activity. Although TMEM16K is widely distributed and associated with the neurological disorder autosomal recessive spinocerebellar ataxia type 10 (SCAR10), its location in cells, function and structure are largely uncharacterised. Here we show that TMEM16K is an ER-resident lipid scramblase with a requirement for short chain lipids and calcium for robust activity. Crystal structures of TMEM16K show a scramblase fold, with an open lipid transporting groove. Additional cryo-EM structures reveal extensive conformational changes from the cytoplasmic to the ER side of the membrane, giving a state with a closed lipid permeation pathway. Molecular dynamics simulations showed that the open-groove conformation is necessary for scramblase activity.
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Sep 2019
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I03-Macromolecular Crystallography
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Marcus
Schewe
,
Han
Sun
,
Ümit
Mert
,
Alexandra
Mackenzie
,
Ashley C. W.
Pike
,
Friederike
Schulz
,
Cristina
Constantin
,
Kirsty S.
Vowinkel
,
Linus J.
Conrad
,
Aytug K.
Kiper
,
Wendy
Gonzalez
,
Marianne
Musinszki
,
Marie
Tegtmeier
,
David C.
Pryde
,
Hassane
Belabed
,
Marc
Nazare
,
Bert L.
De Groot
,
Niels
Decher
,
Bernd
Fakler
,
Elisabeth P.
Carpenter
,
Stephen J.
Tucker
,
Thomas
Baukrowitz
Diamond Proposal Number(s):
[8421]
Abstract: Potassium (K+) channels have been evolutionarily tuned for activation by diverse biological stimuli, and pharmacological activation is thought to target these specific gating mechanisms. Here we report a class of negatively charged activators (NCAs) that bypass the specific mechanisms but act as master keys to open K+ channels gated at their selectivity filter (SF), including many two-pore domain K+ (K2P) channels, voltage-gated hERG (human ether-à-go-go–related gene) channels and calcium (Ca2+)–activated big-conductance potassium (BK)–type channels. Functional analysis, x-ray crystallography, and molecular dynamics simulations revealed that the NCAs bind to similar sites below the SF, increase pore and SF K+ occupancy, and open the filter gate. These results uncover an unrecognized polypharmacology among K+ channel activators and highlight a filter gating machinery that is conserved across different families of K+ channels with implications for rational drug design.
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Feb 2019
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Yin Yao
Dong
,
Hua
Wang
,
Ashley C. W.
Pike
,
Stephen A.
Cochrane
,
Sadra
Hamedzadeh
,
Filip J.
Wyszyński
,
Simon R.
Bushell
,
Sylvain F.
Royer
,
David A.
Widdick
,
Andaleeb
Sajid
,
Helena I.
Boshoff
,
Yumi
Park
,
Ricardo
Lucas
,
Wei-Min
Liu
,
Seung Seo
Lee
,
Takuya
Machida
,
Leanne
Minall
,
Shahid
Mehmood
,
Katsiaryna
Belaya
,
Wei-Wei
Liu
,
Amy
Chu
,
Leela
Shrestha
,
Shubhashish M. M.
Mukhopadhyay
,
Claire
Strain-Damerell
,
Rod
Chalk
,
Nicola A.
Burgess-Brown
,
Mervyn J.
Bibb
,
Clifton E.
Barry
,
Carol V.
Robinson
,
David
Beeson
,
Benjamin G.
Davis
,
Elizabeth P.
Carpenter
Diamond Proposal Number(s):
[10619, 15433, 19301]
Open Access
Abstract: Protein N-glycosylation is a widespread post-translational modification. The first committed step in this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 (GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (thus causing disease) and allow design of non-toxic ‘lipid-altered’ tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed the design of potent antibiotics for Mycobacterium tuberculosis, enabling treatment in vitro, in cellulo and in vivo, providing a promising new class of antimicrobial drug.
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Nov 2018
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