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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I04-1-Macromolecular Crystallography (fixed wavelength)
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Anthony R.
Bradley
,
Aude
Echalier
,
Michael
Fairhead
,
Claire
Strain-Damerell
,
Paul
Brennan
,
Alex n.
Bullock
,
Nicola a.
Burgess-Brown
,
Elizabeth P.
Carpenter
,
Opher
Gileadi
,
Brian d.
Marsden
,
Wen hwa
Lee
,
Wyatt
Yue
,
Chas
Bountra
,
Frank
Von Delft
Open Access
Abstract: The ongoing explosion in genomics data has long since outpaced the capacity of conventional biochemical methodology to verify the large number of hypotheses that emerge from the analysis of such data. In contrast, it is still a gold-standard for early phenotypic validation towards small-molecule drug discovery to use probe molecules (or tool compounds), notwithstanding the difficulty and cost of generating them. Rational structure-based approaches to ligand discovery have long promised the efficiencies needed to close this divergence; in practice, however, this promise remains largely unfulfilled, for a host of well-rehearsed reasons and despite the huge technical advances spearheaded by the structural genomics initiatives of the noughties. Therefore the current, fourth funding phase of the Structural Genomics Consortium (SGC), building on its extensive experience in structural biology of novel targets and design of protein inhibitors, seeks to redefine what it means to do structural biology for drug discovery. We developed the concept of a Target Enabling Package (TEP) that provides, through reagents, assays and data, the missing link between genetic disease linkage and the development of usefully potent compounds. There are multiple prongs to the ambition: rigorously assessing targets’ genetic disease linkages through crowdsourcing to a network of collaborating experts; establishing a systematic approach to generate the protocols and data that comprise each target’s TEP; developing new, X-ray-based fragment technologies for generating high quality chemical matter quickly and cheaply; and exploiting a stringently open access model to build multidisciplinary partnerships throughout academia and industry. By learning how to scale these approaches, the SGC aims to make structures finally serve genomics, as originally intended, and demonstrate how 3D structures systematically allow new modes of druggability to be discovered for whole classes of targets.
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Nov 2017
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Mariana
Grieben
,
Ashley C. W.
Pike
,
Chitra A.
Shintre
,
Elisa
Venturi
,
Sam
El-Ajouz
,
Annamaria
Tessitore
,
Leela
Shrestha
,
Shubhashish
Mukhopadhyay
,
Pravin
Mahajan
,
Rod
Chalk
,
Nicola A
Burgess-Brown
,
Rebecca
Sitsapesan
,
Juha T.
Huiskonen
,
Elisabeth P.
Carpenter
Diamond Proposal Number(s):
[10619]
Abstract: Mutations in either polycystin-1 (PC1 or PKD1) or polycystin-2 (PC2, PKD2 or TRPP1) cause autosomal-dominant polycystic kidney disease (ADPKD) through unknown mechanisms. Here we present the structure of human PC2 in a closed conformation, solved by electron cryomicroscopy at 4.2-Å resolution. The structure reveals a novel polycystin-specific 'tetragonal opening for polycystins' (TOP) domain tightly bound to the top of a classic transient receptor potential (TRP) channel structure. The TOP domain is formed from two extensions to the voltage-sensor-like domain (VSLD); it covers the channel's endoplasmic reticulum lumen or extracellular surface and encloses an upper vestibule, above the pore filter, without blocking the ion-conduction pathway. The TOP-domain fold is conserved among the polycystins, including the homologous channel-like region of PC1, and is the site of a cluster of ADPKD-associated missense variants. Extensive contacts among the TOP-domain subunits, the pore and the VSLD provide ample scope for regulation through physical and chemical stimuli.
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Dec 2016
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|
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Open Access
Abstract: Heavy-atom derivatization is one of the oldest techniques for obtaining phase information for protein crystals and, although it is no longer the first choice, it remains a useful technique for obtaining phases for unknown structures and for low-resolution data sets. It is also valuable for confirming the chain trace in low-resolution electron-density maps. This overview provides a summary of the technique and is aimed at first-time users of the method. It includes guidelines on when to use it, which heavy atoms are most likely to work, how to prepare heavy-atom solutions, how to derivatize crystals and how to determine whether a crystal is in fact a derivative.
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Mar 2016
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I02-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Y. Y.
Dong
,
A.
Pike
,
A.
Mackenzie
,
C.
Mcclenaghan
,
P.
Aryal
,
L.
Dong
,
A.
Quigley
,
M.
Grieben
,
S.
Goubin
,
S.
Mukhopadhyay
,
G. F.
Ruda
,
M. V.
Clausen
,
L.
Cao
,
P. E.
Brennan
,
N. A.
Burgess-Brown
,
M. S. P.
Sansom
,
S. J.
Tucker
,
E. P.
Carpenter
Diamond Proposal Number(s):
[8421, 10619]
Abstract: TREK-2 (KCNK10/K2P10), a two-pore domain potassium (K2P) channel, is gated by multiple stimuli such as stretch, fatty acids, and pH and by several drugs. However, the mechanisms that control channel gating are unclear. Here we present crystal structures of the human TREK-2 channel (up to 3.4 angstrom resolution) in two conformations and in complex with norfluoxetine, the active metabolite of fluoxetine (Prozac) and a state-dependent blocker of TREK channels. Norfluoxetine binds within intramembrane fenestrations found in only one of these two conformations. Channel activation by arachidonic acid and mechanical stretch involves conversion between these states through movement of the pore-lining helices. These results provide an explanation for TREK channel mechanosensitivity, regulation by diverse stimuli, and possible off-target effects of the serotonin reuptake inhibitor Prozac.
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Mar 2015
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