I24-Microfocus Macromolecular Crystallography
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Yang
Yang
,
Hye Jin
Kang
,
Ruogu
Gao
,
Jingjing
Wang
,
Gye Won
Han
,
Jeffrey F.
Diberto
,
Lijie
Wu
,
Jiahui
Tong
,
Lu
Qu
,
Yiran
Wu
,
Ryan
Pileski
,
Xuemei
Li
,
Xuejun Cai
Zhang
,
Suwen
Zhao
,
Terry
Kenakin
,
Quan
Wang
,
Raymond C.
Stevens
,
Wei
Peng
,
Bryan L.
Roth
,
Zihe
Rao
,
Zhi-Jie
Liu
Open Access
Abstract: The hydroxycarboxylic acid receptor 2 (HCA2) agonist niacin has been used as treatment for dyslipidemia for several decades albeit with skin flushing as a common side-effect in treated individuals. Extensive efforts have been made to identify HCA2 targeting lipid lowering agents with fewer adverse effects, despite little being known about the molecular basis of HCA2 mediated signalling. Here, we report the cryo-electron microscopy structure of the HCA2-Gi signalling complex with the potent agonist MK-6892, along with crystal structures of HCA2 in inactive state. These structures, together with comprehensive pharmacological analysis, reveal the ligand binding mode and activation and signalling mechanisms of HCA2. This study elucidates the structural determinants essential for HCA2 mediated signalling and provides insights into ligand discovery for HCA2 and related receptors.
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Mar 2023
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Abstract: Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a life-threatening disease that has plagued humanity for thousands of years. Mycobacteria, including Mtb, synthesise a complex cell wall to support and protect the bacterial cells. The front-line anti-TB drug ethambutol inhibits the synthesis of the mycobacterial cell wall by targeting the Emb proteins (EmbA, EmbB and EmbC). However, although the drug has been in use for fifty years, its mode of action remains unclear.
InMtb,EmbCisrequiredforthesynthesisoflipoarabinomannan(LAM),animportantvirulencefactorthataddstothepathogen’seffectiveness and helps it infect host cells. LAM also plays a crucial role in host-pathogen interactions and modulating the host immune response during infection. However, the three-dimensional structure of the Emb proteins had yet to be determined. To address the lack of structural information and functional analysis, an international team of researchers determined the crystal structure of EmbC from Mycobacterium smegmatis in complex with a sugar substrate, using Macromolecular Crystallography (MX) on Diamond Light Source's I04-1 beamline.
Unexpectedly, they found that the mycobacterial acyl-carrier-protein (AcpM) is bound to EmbC to form a stable EmbC2-AcpM2 complex. A combination of the MX data with a cryo-EM structure of the ethambutol-bound EmbC2-AcpM2 complex suggests that ethambutol works by binding to the same sites as both donor and acceptor substrates of EmbC. These results provide a structural basis for understanding the biochemical function and inhibition of EmbC and the development of new anti-tuberculosis agents.
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Jul 2021
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I04-Macromolecular Crystallography
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Kangsa
Amporndanai
,
Xiaoli
Meng
,
Weijuan
Shang
,
Zhenmig
Jin
,
Michael
Rogers
,
Yao
Zhao
,
Zihe
Rao
,
Zhi-Jie
Liu
,
Haitao
Yang
,
Leike
Zhang
,
Paul M.
O’neill
,
S. Samar
Hasnain
Diamond Proposal Number(s):
[27113]
Open Access
Abstract: The SARS-CoV-2 pandemic has triggered global efforts to develop therapeutics. The main protease of SARS-CoV-2 (Mpro), critical for viral replication, is a key target for therapeutic development. An organoselenium drug called ebselen has been demonstrated to have potent Mpro inhibition and antiviral activity. We have examined the binding modes of ebselen and its derivative in Mpro via high resolution co-crystallography and investigated their chemical reactivity via mass spectrometry. Stronger Mpro inhibition than ebselen and potent ability to rescue infected cells were observed for a number of derivatives. A free selenium atom bound with cysteine of catalytic dyad has been revealed in crystallographic structures of Mpro with ebselen and MR6-31-2 suggesting hydrolysis of the enzyme bound organoselenium covalent adduct and formation of a phenolic by-product, confirmed by mass spectrometry. The target engagement with selenation mechanism of inhibition suggests wider therapeutic applications of these compounds against SARS-CoV-2 and other zoonotic beta-corona viruses.
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May 2021
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Tika R.
Malla
,
Anthony
Tumber
,
Tobias
John
,
Lennart
Brewitz
,
Claire
Strain-Damerell
,
C. David
Owen
,
Petra
Lukacik
,
H. T. Henry
Chan
,
Pratheesh
Maheswaran
,
Eidarus
Salah
,
Fernanda
Duarte
,
Haitao
Yang
,
Zihe
Rao
,
Martin A.
Walsh
,
Christopher J.
Schofield
Open Access
Abstract: The main viral protease (Mpro) of SARS-CoV-2 is a nucleophilic cysteine hydrolase and a current target for anti-viral chemotherapy. We describe a high-throughput solid phase extraction coupled to mass spectrometry Mpro assay. The results reveal some β-lactams, including penicillin esters, are active site reacting Mpro inhibitors, thus highlighting the potential of acylating agents for Mpro inhibition.
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Jan 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Lu
Zhang
,
Yao
Zhao
,
Yan
Gao
,
Lijie
Wu
,
Ruogu
Gao
,
Qi
Zhang
,
Yinan
Wang
,
Chengyao
Wu
,
Fangyu
Wu
,
Sudagar S.
Gurcha
,
Natacha
Veerapen
,
Sarah M.
Batt
,
Wei
Zhao
,
Ling
Qin
,
Xiuna
Yang
,
Manfu
Wang
,
Yan
Zhu
,
Bing
Zhang
,
Lijun
Bi
,
Xian’en
Zhang
,
Haitao
Yang
,
Luke W.
Guddat
,
Wenqing
Xu
,
Quan
Wang
,
Jun
Li
,
Gurdyal S.
Besra
,
Zihe
Rao
Abstract: The arabinosyltransferases EmbA, EmbB, and EmbC are involved in Mycobacterium tuberculosis cell wall synthesis and are recognized as the targets for the anti-tuberculosis drug ethambutol. We have determined cryo-electron microscopy and x-ray crystal structures of mycobacterial EmbA-EmbB and EmbC-EmbC complexes, in the presence of their glycosyl donor and acceptor substrates and with ethambutol. These structures show how the donor and acceptor substrates bind in the active site and how ethambutol inhibits by binding to the same site as both substrates in EmbB and EmbC. The majority of drug-resistant mutations are located nearby to the ethambutol-binding site. Collectively, our work provides a structural basis for understanding the biochemical function and inhibition of arabinosyltransferases and development of new anti-tuberculosis agents.
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Apr 2020
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Yuguang
Zhao
,
Daming
Zhou
,
Tao
Ni
,
Dimple
Karia
,
Abhay
Kotecha
,
Xiangxi
Wang
,
Zihe
Rao
,
E. Yvonne
Jones
,
Elizabeth E.
Fry
,
Jingshan
Ren
,
David I.
Stuart
Open Access
Abstract: Coxsackievirus A10 (CV-A10) is responsible for an escalating number of severe infections in children, but no prophylactics or therapeutics are currently available. KREMEN1 (KRM1) is the entry receptor for the largest receptor-group of hand-foot-and-mouth disease causing viruses, which includes CV-A10. We report here structures of CV-A10 mature virus alone and in complex with KRM1 as well as of the CV-A10 A-particle. The receptor spans the viral canyon with a large footprint on the virus surface. The footprint has some overlap with that seen for the neonatal Fc receptor complexed with enterovirus E6 but is larger and distinct from that of another enterovirus receptor SCARB2. Reduced occupancy of a particle-stabilising pocket factor in the complexed virus and the presence of both unbound and expanded virus particles suggests receptor binding initiates a cascade of conformational changes that produces expanded particles primed for viral uncoating.
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Jan 2020
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Shaowei
Zhang
,
Derren J.
Heyes
,
Lingling
Feng
,
Wenli
Sun
,
Linus O.
Johannissen
,
Huanting
Liu
,
Colin
Levy
,
Xuemei
Li
,
Ji
Yang
,
Xiaolan
Yu
,
Min
Lin
,
Samantha J. O.
Hardman
,
Robin
Hoeven
,
Michiyo
Sakuma
,
Sam
Hay
,
David
Leys
,
Zihe
Rao
,
Aiwu
Zhou
,
Qi
Cheng
,
Nigel S.
Scrutton
Diamond Proposal Number(s):
[8997, 12788]
Abstract: The enzyme protochlorophyllide oxidoreductase (POR) catalyses a light-dependent step in chlorophyll biosynthesis that is essential to photosynthesis and, ultimately, all life on Earth1,2,3. POR, which is one of three known light-dependent enzymes4,5, catalyses reduction of the photosensitizer and substrate protochlorophyllide to form the pigment chlorophyllide. Despite its biological importance, the structural basis for POR photocatalysis has remained unknown. Here we report crystal structures of cyanobacterial PORs from Thermosynechococcus elongatus and Synechocystis sp. in their free forms, and in complex with the nicotinamide coenzyme. Our structural models and simulations of the ternary protochlorophyllide–NADPH–POR complex identify multiple interactions in the POR active site that are important for protochlorophyllide binding, photosensitization and photochemical conversion to chlorophyllide. We demonstrate the importance of active-site architecture and protochlorophyllide structure in driving POR photochemistry in experiments using POR variants and protochlorophyllide analogues. These studies reveal how the POR active site facilitates light-driven reduction of protochlorophyllide by localized hydride transfer from NADPH and long-range proton transfer along structurally defined proton-transfer pathways.
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Oct 2019
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Bing
Zhang
,
Jun
Li
,
Xiaolin
Yang
,
Lijie
Wu
,
Jia
Zhang
,
Yang
Yang
,
Yao
Zhao
,
Lu
Zhang
,
Xiuna
Yang
,
Xiaobao
Yang
,
Xi
Cheng
,
Zhijie
Liu
,
Biao
Jiang
,
Hualiang
Jiang
,
Luke W.
Guddat
,
Haitao
Yang
,
Zihe
Rao
Abstract: Despite intensive efforts to discover highly effective treatments to eradicate tuberculosis (TB), it remains as a major threat to global human health. For this reason, new TB drugs directed toward new targets are highly coveted. MmpLs (Mycobacterial membrane proteins Large), which play crucial roles in transporting lipids, polymers and immunomodulators and which also extrude therapeutic drugs, are among the most important therapeutic drug targets to emerge in recent times. Here, crystal structures of mycobacterial MmpL3 alone and in complex with four TB drug candidates, including SQ109 (in Phase 2b-3 clinical trials), are reported. MmpL3 consists of a periplasmic pore domain and a twelve-helix transmembrane domain. Two Asp-Tyr pairs centrally located in this domain appear to be key facilitators of proton-translocation. SQ109, AU1235, ICA38, and rimonabant bind inside the transmembrane region and disrupt these Asp-Tyr pairs. This structural data will greatly advance the development of MmpL3 inhibitors as new TB drugs.
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Jan 2019
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Abstract: Enterovirus 71 (EV71) is a common cause of hand, foot and mouth disease—a disease endemic especially in the Asia-Pacific region1. Scavenger receptor class B member 2 (SCARB2) is the major receptor of EV71, as well as several other enteroviruses responsible for hand, foot and mouth disease, and plays a key role in cell entry2. The isolated structures of EV71 and SCARB2 are known3,4,5,6, but how they interact to initiate infection is not. Here, we report the EV71–SCARB2 complex structure determined at 3.4 Å resolution using cryo-electron microscopy. This reveals that SCARB2 binds EV71 on the southern rim of the canyon, rather than across the canyon, as predicted3,7,8. Helices 152–163 (α5) and 183–193 (α7) of SCARB2 and the viral protein 1 (VP1) GH and VP2 EF loops of EV71 dominate the interaction, suggesting an allosteric mechanism by which receptor binding might facilitate the low-pH uncoating of the virus in the endosome/lysosome. Remarkably, many residues within the binding footprint are not conserved across SCARB2-dependent enteroviruses; however, a conserved proline and glycine seem to be key residues. Thus, although the virus maintains antigenic variability even within the receptor-binding footprint, the identification of binding ‘hot spots’ may facilitate the design of receptor mimic therapeutics less likely to quickly generate resistance.
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Dec 2018
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[6387, 8423]
Abstract: Hepatitis A virus (HAV) has been enigmatic, evading detailed structural analysis for many years. Its recently determined high-resolution structure revealed an angular surface without the indentations often characteristic of receptor-binding sites. The viral protein 1 (VP1) β-barrel shows no sign of a pocket factor and the amino terminus of VP2 displays a “domain swap” across the pentamer interface, as in a subset of mammalian picornaviruses and insect picorna-like viruses. Structure-based phylogeny confirms this placement. These differences suggest an uncoating mechanism distinct from that of enteroviruses. An empty capsid structure reveals internal differences in VP0 and the VP1 amino terminus connected with particle maturation. An HAV/Fab complex structure, in which the antigen-binding fragment (Fab) appears to act as a receptor–mimic, clarifies some historical epitope mapping data, but some remain difficult to reconcile. We still have little idea of the structural features of enveloped HAV particles.
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Jul 2018
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