I04-Macromolecular Crystallography
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Linxiang
Yin
,
Geoffrey
Masuyer
,
Sicai
Zhang
,
Jie
Zhang
,
Shin-ichiro
Miyashita
,
David
Burgin
,
Laura
Lovelock
,
Shu-fen
Coker
,
Tian-min
Fu
,
Pal
Stenmark
,
Min
Dong
Diamond Proposal Number(s):
[15806]
Open Access
Abstract: Botulinum neurotoxins (BoNTs) are a family of bacterial toxins with seven major serotypes (BoNT/A-G). The ability of these toxins to target and bind to motor nerve terminals is a key factor determining their potency and efficacy. Among these toxins, BoNT/B is one of the two types approved for medical and cosmetic uses. Besides binding to well-established receptors, an extended loop in the C-terminal receptor-binding domain (HC) of BoNT/B (HC/B) has been proposed to also contribute to toxin binding to neurons by interacting with lipid membranes (termed lipid-binding loop [LBL]). Analogous loops exist in the HCs of BoNT/C, D, G, and a chimeric toxin DC. However, it has been challenging to detect and characterize binding of LBLs to lipid membranes. Here, using the nanodisc system and biolayer interferometry assays, we find that HC/DC, C, and G, but not HC/B and HC/D, are capable of binding to receptor-free lipids directly, with HC/DC having the highest level of binding. Mutagenesis studies demonstrate the critical role of consecutive aromatic residues at the tip of the LBL for binding of HC/DC to lipid membranes. Taking advantage of this insight, we then create a "gain-of-function" mutant HC/B by replacing two nonaromatic residues at the tip of its LBL with tryptophan. Cocrystallization studies confirm that these two tryptophan residues do not alter the structure of HC/B or the interactions with its receptors. Such a mutated HC/B gains the ability to bind receptor-free lipid membranes and shows enhanced binding to cultured neurons. Finally, full-length BoNT/B containing two tryptophan mutations in its LBL, together with two additional mutations (E1191M/S1199Y) that increase binding to human receptors, is produced and evaluated in mice in vivo using Digit Abduction Score assays. This mutant toxin shows enhanced efficacy in paralyzing local muscles at the injection site and lower systemic diffusion, thus extending both safety range and duration of paralysis compared with the control BoNT/B. These findings establish a mechanistic understanding of LBL-lipid interactions and create a modified BoNT/B with improved therapeutic efficacy.
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Mar 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[11265]
Abstract: Assembly of the mitochondrial respiratory chain requires the coordinated synthesis of mitochondrial and nuclear encoded subunits, redox co-factor acquisition, and correct joining of the subunits to form functional complexes. The conserved Cbp3–Cbp6 chaperone complex binds newly synthesized cytochrome b and supports the ordered acquisition of the heme co-factors. Moreover, it functions as a translational activator by interacting with the mitoribosome. Cbp3 consists of two distinct domains, an N-terminal domain present in mitochondrial Cbp3 homologs, and a highly conserved C-terminal domain comprising a ubiquinol–cytochrome c chaperone region. Here, we solved the crystal structure of this C-terminal domain from a bacterial homolog at 1.4 Å resolution, revealing a unique all-helical fold. This structure allowed mapping of the interaction sites of yeast Cbp3 with Cbp6 and cytochrome b via site-specific photo-crosslinking. We propose that mitochondrial Cbp3 homologs carry an N-terminal extension that positions the conserved C-terminal domain at the ribosomal tunnel exit for an efficient interaction with its substrate, the newly synthesized cytochrome b protein.
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Sep 2019
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[15806]
Abstract: Botulinum neurotoxins (BoNTs) are the most potent toxins known. So far, eight serotypes have been identified that all act as zinc‐dependent endopeptidases targeting SNARE proteins and inhibiting the release of neurotransmitters. Recently, the first botulinum toxin‐like protein was identified outside the Clostridial genus, designated BoNT/Wo in the genome of Weissella oryzae. Here, we report the 1.6 Å X‐ray crystal structure of the light chain of BoNT/Wo (LC/Wo). LC/Wo presents the core fold common to BoNTs but has an unusually wide, open, and negatively charged catalytic pocket, with an additional Ca2+ ion besides the zinc ion and a unique ß‐hairpin motif. The structural information will help establish the substrate profile of BoNT/Wo and help our understanding of how BoNT evolved.
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May 2019
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Mark
Elliott
,
Christine
Favre-guilmard
,
Sai Man
Liu
,
Jacquie
Maignel
,
Geoffrey
Masuyer
,
Matthew
Beard
,
Christopher
Boone
,
Denis
Carré
,
Mikhail
Kalinichev
,
Stephane
Lezmi
,
Imran
Mir
,
Camille
Nicoleau
,
Shilpa
Palan
,
Cindy
Perier
,
Elsa
Raban
,
Sicai
Zhang
,
Min
Dong
,
Pal
Stenmark
,
Johannes
Krupp
Diamond Proposal Number(s):
[11265, 15806]
Open Access
Abstract: Although botulinum neurotoxin serotype A (BoNT/A) products are common treatments for various disorders, there is only one commercial BoNT/B product, whose low potency, likely stemming from low affinity toward its human receptor synaptotagmin 2 (hSyt2), has limited its therapeutic usefulness. We express and characterize two full-length recombinant BoNT/B1 proteins containing designed mutations E1191M/S1199Y (rBoNT/B1MY) and E1191Q/S1199W (rBoNT/B1QW) that enhance binding to hSyt2. In preclinical models including human-induced pluripotent stem cell neurons and a humanized transgenic mouse, this increased hSyt2 affinity results in high potency, comparable to that of BoNT/A. Last, we solve the cocrystal structure of rBoNT/B1MY in complex with peptides of hSyt2 and its homolog hSyt1. We demonstrate that neuronal surface receptor binding limits the clinical efficacy of unmodified BoNT/B and that modified BoNT/B proteins have promising clinical potential.
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Jan 2019
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Torkild
Visnes
,
Armando
Cázares-körner
,
Wenjing
Hao
,
Olov
Wallner
,
Geoffrey
Masuyer
,
Olga
Loseva
,
Oliver
Mortusewicz
,
Elisée
Wiita
,
Antonio
Sarno
,
Aleksandr
Manoilov
,
Juan
Astorga-wells
,
Ann-sofie
Jemth
,
Lang
Pan
,
Kumar
Sanjiv
,
Stella
Karsten
,
Camilla
Gokturk
,
Maurice
Grube
,
Evert J.
Homan
,
Bishoy M. F.
Hanna
,
Cynthia B. J.
Paulin
,
Therese
Pham
,
Azita
Rasti
,
Ulrika Warpman
Berglund
,
Catharina
Von Nicolai
,
Carlos
Benitez-buelga
,
Tobias
Koolmeister
,
Dag
Ivanic
,
Petar
Iliev
,
Martin
Scobie
,
Hans E.
Krokan
,
Pawel
Baranczewski
,
Per
Artursson
,
Mikael
Altun
,
Annika Jenmalm
Jensen
,
Christina
Kalderén
,
Xueqing
Ba
,
Roman A.
Zubarev
,
Pal
Stenmark
,
Istvan
Boldogh
,
Thomas
Helleday
Diamond Proposal Number(s):
[15806]
Abstract: The onset of inflammation is associated with reactive oxygen species and oxidative damage to macromolecules like 7,8-dihydro-8-oxoguanine (8-oxoG) in DNA. Because 8-oxoguanine DNA glycosylase 1 (OGG1) binds 8-oxoG and because Ogg1-deficient mice are resistant to acute and systemic inflammation, we hypothesized that OGG1 inhibition may represent a strategy for the prevention and treatment of inflammation. We developed TH5487, a selective active-site inhibitor of OGG1, which hampers OGG1 binding to and repair of 8-oxoG and which is well tolerated by mice. TH5487 prevents tumor necrosis factor–α–induced OGG1-DNA interactions at guanine-rich promoters of proinflammatory genes. This, in turn, decreases DNA occupancy of nuclear factor κB and proinflammatory gene expression, resulting in decreased immune cell recruitment to mouse lungs. Thus, we present a proof of concept that targeting oxidative DNA repair can alleviate inflammatory conditions in vivo.
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Nov 2018
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[11265]
Open Access
Abstract: Botulinum neurotoxins (BoNTs) are among the most potent toxins known and are also used to treat an increasing number of medical disorders. There are seven well-established serotypes (BoNT/A-G), which all act as zinc-dependent endopeptidases targeting specific members of the SNARE proteins required for synaptic vesicle exocytosis in neurons. A new toxin serotype, BoNT/X, was recently identified. It cleaves not only the canonical targets, vesicle associated membrane proteins (VAMP) 1/2/3 at a unique site, but also has the unique ability to cleave VAMP4/5 and Ykt6. Here we report the 1.35 Å X-ray crystal structure of the light chain of BoNT/X (LC/X). LC/X shares the core fold common to all other BoNTs, demonstrating that LC/X is a bona fide member of BoNT-LCs. We found that access to the catalytic pocket of LC/X is more restricted, and the regions lining the catalytic pocket are not conserved compared to other BoNTs. Kinetic studies revealed that LC/X cleaves VAMP1 with a ten times higher efficiency than BoNT/B and the tetanus neurotoxin. The structural information provides a molecular basis to understand the convergence/divergence between BoNT/X and other BoNTs, to develop effective LC inhibitors, and to engineer new scientific tools and therapeutic toxins targeting distinct SNARE proteins in cells.
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Mar 2018
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B21-High Throughput SAXS
I04-1-Macromolecular Crystallography (fixed wavelength)
Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[11265]
Abstract: The tetanus neurotoxin (TeNT) is a highly potent toxin produced by Clostridium tetani that inhibits neurotransmission of inhibitory interneurons, causing spastic paralysis in the tetanus disease. TeNT differs from the other clostridial neurotoxins by its unique ability to target the central nervous system by retrograde axonal transport. The crystal structure of the tetanus toxin reveals a “closed” domain arrangement stabilised by two disulphide bridges, and the molecular details of the toxin's interaction with its polysaccharide receptor. An integrative analysis combining X-ray crystallography, solution scattering and single particle electron cryo-microscopy reveals pH-mediated domain rearrangements that may give TeNT the ability to adapt to the multiple environments encountered during intoxication, and facilitate binding to distinct receptors.
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Jun 2017
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8922]
Open Access
Abstract: Several soil-derived Actinobacteria produce secondary metabolites that are proven specific and potent inhibitors of the human angiotensin-I-converting enzyme (ACE), a key target for the modulation of hypertension through its role in the renin–angiotensin–aldosterone system. K-26-DCP is a zinc dipeptidyl carboxypeptidase (DCP) produced by Astrosporangium hypotensionis, and an ancestral homologue of ACE. Here we report the high-resolution crystal structures of K-26-DCP and of its complex with the natural microbial tripeptide product K-26. The experimental results provide the structural basis for better understanding the specificity of K-26 for human ACE over bacterial DCPs.
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Nov 2016
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I03-Macromolecular Crystallography
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Open Access
Abstract: Angiotensin-1 converting enzyme (ACE), a zinc metallopeptidase, consists of two homologous catalytic domains (N and C) with different substrate specificities. Here we report kinetic parameters of five different forms of human ACE with various amyloid beta (A) substrates together with high resolution crystal structures of N-domain in complex with A fragments. For the physiological A(1-16) peptide, a novel ACE cleavage site was found at His14/Gln15. Furthermore, A(1-16) was preferentially cleaved by the individual N-domain; however, the presence of an inactive C-domain in full-length sACE greatly reduced enzyme activity and affected apparent selectivity. Two fluorogenic substrates, A(4-10)Q and A(4-10)Y underwent endoproteolytic cleavage at the Asp7/Ser8 bond with all ACE constructs showing greater catalytic efficiency for A(4-10)Y. Surprisingly, in contrast to A(1-16) and A(4-10)Q, sACE showed positive domain cooperativity and the double C-domain (CC-sACE) construct no cooperativity towards A(4-10)Y. The structures of the A peptide-ACE complexes revealed a common mode of peptide binding for both domains which principally targets the C-terminal Pβ’ position to the Sβ’ pocket and recognises the main chain of the P1’ peptide. It is likely that N-domain selectivity for the amyloid peptide is conferred through the N-domain specific Sβ’ residue Thrγ58. Additionally, the N-domain can accommodate larger substrates through movement of the N-terminal helices, as suggested by the disorder of the hinge region in the crystal structures. Our findings are important for the design of domain selective inhibitors as the differences in domain-selectivity are more pronounced with the truncated domains compared to the more physiological full-length forms.
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Jan 2016
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8922]
Open Access
Abstract: The botulinum neurotoxin type D is one of seven highly potent toxins produced by Clostridium botulinum which inhibit neurotransmission at cholinergic nerve terminals. A functional fragment derived from the toxin, LHn, consisting of the catalytic and translocation domains, has been heralded as a platform for the development of targeted secretion inhibitors. These secretion inhibitors are aimed at retargeting the toxin towards a specific cell type to inhibit vesicular secretion. Here we report crystal structures of LHn from serotype D at 2.3 Å, and that of SXN101959 at 3.1 Å resolution. SXN101959, a derivative that combines LHn from serotype D with a fragment of the growth hormone releasing hormone, has previously revealed promising results in inhibiting growth hormone release in pituitary somatotrophs. These structures offer for the first time insights into the translocation domain interaction with the catalytic domain in serotype D. Furthermore, structural information from small-angle X-ray scattering of LHn/D is compared among serotypes A, B, and D. Taken together, these results demonstrate the robustness of the ‘LHn fold’ across serotypes and its use in engineering additional polypeptide components with added functionality. Our study demonstrates the suitability of botulinum neurotoxin, and serotype D in particular, as a basis for engineering novel secretion inhibitors.
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Sep 2015
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