I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
|
Lucile
Moynie
,
Françoise
Hoegy
,
Stefan
Milenkovic
,
Mathilde
Munier
,
Aurélie
Paulen
,
Véronique
Gasser
,
Aline L.
Faucon
,
Nicolas
Zill
,
James H.
Naismith
,
Matteo
Ceccarelli
,
Isabelle J.
Schalk
,
Gaëtan L. A.
Mislin
Diamond Proposal Number(s):
[19946, 19281]
Abstract: Enterobactin (ENT) is a tris-catechol siderophore used to acquire iron by multiple bacterial species. These ENT-dependent iron uptake systems have often been considered as potential gates in the bacterial envelope through which one can shuttle antibiotics (Trojan horse strategy). In practice, siderophore analogues containing catechol moieties have shown promise as vectors to which antibiotics may be attached. Bis- and tris-catechol vectors (BCVs and TCVs, respectively) were shown using structural biology and molecular modeling to mimic ENT binding to the outer membrane transporter PfeA in Pseudomonas aeruginosa. TCV but not BCV appears to cross the outer membrane via PfeA when linked to an antibiotic (linezolid). TCV is therefore a promising vector for Trojan horse strategies against P. aeruginosa, confirming the ENT-dependent iron uptake system as a gate to transport antibiotics into P. aeruginosa cells.
|
Jul 2022
|
|
I03-Macromolecular Crystallography
Krios II-Titan Krios II at Diamond
Krios IV-Titan Krios IV at Diamond
|
Halina
Mikolajek
,
Miriam
Weckener
,
Z. Faidon
Brotzakis
,
Jiandong
Huo
,
Evmorfia V.
Dalietou
,
Audrey
Le Bas
,
Pietro
Sormanni
,
Peter J.
Harrison
,
Philip N.
Ward
,
Steven
Truong
,
Lucile
Moynie
,
Daniel K.
Clare
,
Maud
Dumoux
,
Joshua
Dormon
,
Chelsea
Norman
,
Naveed
Hussain
,
Vinod
Vogirala
,
Raymond J.
Owens
,
Michele
Vendruscolo
,
James
Naismith
Diamond Proposal Number(s):
[27031, 27051, 29666]
Open Access
Abstract: Camelid single-domain antibodies, also known as nanobodies, can be readily isolated from naïve libraries for specific targets but often bind too weakly to their targets to be immediately useful. Laboratory-based genetic engineering methods to enhance their affinity, termed maturation, can deliver useful reagents for different areas of biology and potentially medicine. Using the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and a naïve library, we generated closely related nanobodies with micromolar to nanomolar binding affinities. By analyzing the structure–activity relationship using X-ray crystallography, cryoelectron microscopy, and biophysical methods, we observed that higher conformational entropy losses in the formation of the spike protein–nanobody complex are associated with tighter binding. To investigate this, we generated structural ensembles of the different complexes from electron microscopy maps and correlated the conformational fluctuations with binding affinity. This insight guided the engineering of a nanobody with improved affinity for the spike protein.
|
Jul 2022
|
|
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Jiandong
Huo
,
Halina
Mikolajek
,
Audrey
Le Bas
,
Jordan J.
Clark
,
Parul
Sharma
,
Anja
Kipar
,
Joshua
Dormon
,
Chelsea
Norman
,
Miriam
Weckener
,
Daniel K.
Clare
,
Peter J.
Harrison
,
Julia A.
Tree
,
Karen R.
Buttigieg
,
Francisco J.
Salguero
,
Robert
Watson
,
Daniel
Knott
,
Oliver
Carnell
,
Didier
Ngabo
,
Michael J.
Elmore
,
Susan
Fotheringham
,
Adam
Harding
,
Lucile
Moynie
,
Philip N.
Ward
,
Maud
Dumoux
,
Tessa
Prince
,
Yper
Hall
,
Julian A.
Hiscox
,
Andrew
Owen
,
William
James
,
Miles W.
Carroll
,
James P.
Stewart
,
James
Naismith
,
Raymond
Owens
Diamond Proposal Number(s):
[27031]
Open Access
Abstract: SARS-CoV-2 remains a global threat to human health particularly as escape mutants emerge. There is an unmet need for effective treatments against COVID-19 for which neutralizing single domain antibodies (nanobodies) have significant potential. Their small size and stability mean that nanobodies are compatible with respiratory administration. We report four nanobodies (C5, H3, C1, F2) engineered as homotrimers with pmolar affinity for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Crystal structures show C5 and H3 overlap the ACE2 epitope, whilst C1 and F2 bind to a different epitope. Cryo Electron Microscopy shows C5 binding results in an all down arrangement of the Spike protein. C1, H3 and C5 all neutralize the Victoria strain, and the highly transmissible Alpha (B.1.1.7 first identified in Kent, UK) strain and C1 also neutralizes the Beta (B.1.35, first identified in South Africa). Administration of C5-trimer via the respiratory route showed potent therapeutic efficacy in the Syrian hamster model of COVID-19 and separately, effective prophylaxis. The molecule was similarly potent by intraperitoneal injection.
|
Sep 2021
|
|
I03-Macromolecular Crystallography
Krios I-Titan Krios I at Diamond
|
Jiangdong
Huo
,
Audrey
Le Bas
,
Reinis R.
Ruza
,
Helen M. E.
Duyvesteyn
,
Halina
Mikolajek
,
Tomas
Malinauskas
,
Tiong Kit
Tan
,
Pramila
Rijal
,
Maud
Dumoux
,
Philip N.
Ward
,
Jingshan
Ren
,
Daming
Zhou
,
Peter J.
Harrison
,
Miriam
Weckener
,
Daniel K.
Clare
,
Vinod K.
Vogirala
,
Julika
Radecke
,
Lucile
Moynie
,
Yuguang
Zhao
,
Javier
Gilbert-Jaramillo
,
Michael L.
Knight
,
Julia A.
Tree
,
Karen R.
Buttigieg
,
Naomi
Coombes
,
Michael J.
Elmore
,
Miles W.
Carroll
,
Loic
Carrique
,
Pranav N. M.
Shah
,
William
James
,
Alain R.
Townsend
,
David I.
Stuart
,
Raymond J.
Owens
,
James H.
Naismith
Diamond Proposal Number(s):
[27031, 27051]
Open Access
Abstract: The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than influenza. The SARS-CoV-2 receptor binding domain (RBD) of the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) receptor as a prelude to viral entry into the cell. Using a naive llama single-domain antibody library and PCR-based maturation, we have produced two closely related nanobodies, H11-D4 and H11-H4, that bind RBD (KD of 39 and 12 nM, respectively) and block its interaction with ACE2. Single-particle cryo-EM revealed that both nanobodies bind to all three RBDs in the spike trimer. Crystal structures of each nanobody–RBD complex revealed how both nanobodies recognize the same epitope, which partly overlaps with the ACE2 binding surface, explaining the blocking of the RBD–ACE2 interaction. Nanobody-Fc fusions showed neutralizing activity against SARS-CoV-2 (4–6 nM for H11-H4, 18 nM for H11-D4) and additive neutralization with the SARS-CoV-1/2 antibody CR3022.
|
Jul 2020
|
|
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Lucile
Moynie
,
Stefan
Milenkovic
,
Gaëtan L. A.
Mislin
,
Véronique
Gasser
,
Giuliano
Malloci
,
Etienne
Baco
,
Rory P.
Mccaughan
,
Malcolm G. P.
Page
,
Isabelle J.
Schalk
,
Matteo
Ceccarelli
,
James H.
Naismith
Open Access
Abstract: Bacteria use small molecules called siderophores to scavenge iron. Siderophore-Fe3+ complexes are recognised by outer-membrane transporters and imported into the periplasm in a process dependent on the inner-membrane protein TonB. The siderophore enterobactin is secreted by members of the family Enterobacteriaceae, but many other bacteria including Pseudomonas species can use it. Here, we show that the Pseudomonas transporter PfeA recognises enterobactin using extracellular loops distant from the pore. The relevance of this site is supported by in vivo and in vitro analyses. We suggest there is a second binding site deeper inside the structure and propose that correlated changes in hydrogen bonds link binding-induced structural re-arrangements to the structural adjustment of the periplasmic TonB-binding motif.
|
Aug 2019
|
|
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Open Access
Abstract: New strategies are urgently required to develop antibiotics. The siderophore uptake system has attracted considerable attention but rational design of siderophore antibiotic conjugates requires knowledge of recognition by the cognate outer membrane transporter. Acinetobacter baumannii is a serious pathogen, which utilizes (pre)acinetobactin to scavenge iron from the host. We report the structure of the (pre)acinetobactin transporter BauA bound to the siderophore, identifying the structural determinants of recognition. Detailed biophysical analysis confirms that BauA recognises preacinetobactin. We show that acinetobactin is not recognised by the protein thus preacinetobactin is essential for iron uptake. The structure shows and NMR confirms that under physiological conditions a molecule of acinetobactin will bind to two free coordination sites on the iron preacinetobactin complex. The ability to recognise a heterotrimeric iron preacinetobactin acinetobactin complex may rationalize contradictory reports in the literature. These results open new avenues for the design of novel antibiotic conjugates (trojan horse) antibiotics.
|
Dec 2018
|
|
I24-Microfocus Macromolecular Crystallography
|
Abstract: Enterobactin (ENT) is a siderophore (iron-chelating compound) produced by Escherichia coli in order to gain access to iron, an essential nutriment for bacterial growth. ENT is used as an exosiderophore by the opportunistic human pathogen Pseudomonas aeruginosa with transport of ferri-ENT across the bacterial outer membrane by the transporter PfeA. Next to pfeA gene on the chromosome is localized a gene encoding for an esterase, PfeE, whose transcription is regulated, as for pfeA, by the presence of ENT in bacterial environment. Purified PfeE hydrolyzed ferri-ENT into three molecules of 2,3 DHBS (2,3 dihydroxybenzoylserine) still complexed with ferric iron, and complete dissociation of iron from ENT chelating groups was only possible in the presence of both PfeE and an iron reducer, such as DTT. The crystal structure of PfeE and an inactive PfeE mutant complexed with ferri-ENT or a non-hydrolysable ferri-catechol complex allowed identification of the enzyme binding site and the catalytic triad. Finally, cell fractionation and fluorescence microscopy showed periplasmic localization of PfeE in P. aeruginosa cells. Thus, the molecular mechanism of iron release from ENT in P. aeruginosa differs from that previously described in E. coli. In P. aeruginosa, siderophore hydrolysis occurs in the periplasm, with ENT never reaching the bacterial cytoplasm. In E. coli, ferri-ENT crosses the inner membrane via the ABC transporter FepBCD and ferri-ENT is hydrolyzed by the esterase Fes only once it is in the cytoplasm.
|
Aug 2018
|
|
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Abstract: Biotin is an essential vitamin in plants and mammals, functioning as the carbon dioxide carrier within central lipid metabolism. Bacterial pimeloyl-CoA synthetase (BioW) acts as a highly specific substrate-selection gate, ensuring the integrity of the carbon chain in biotin synthesis. BioW catalyzes the condensation of pimelic acid (C7 dicarboxylic acid) with CoASH in an ATP-dependent manner to form pimeloyl-CoA, the first dedicated biotin building block. Multiple structures of Bacillus subtilis BioW together capture all three substrates, as well as the intermediate pimeloyl-adenylate and product pyrophosphate (PPi), indicating that the enzyme uses an internal ruler to select the correct dicarboxylic acid substrate. Both the catalytic mechanism and the surprising stability of the adenylate intermediate were rationalized through site-directed mutagenesis. Building on this understanding, BioW was engineered to synthesize high-value heptanoyl (C7) and octanoyl (C8) monocarboxylic acid-CoA and C8 dicarboxylic-CoA products, highlighting the enzyme's synthetic potential.
|
Apr 2017
|
|
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[14980]
Abstract: The outer membrane of Gram-negative bacteria presents an efficient barrier to the permeation of antimicrobial molecules. One strategy pursued to circumvent this obstacle is to hijack transport systems for essential nutrients, such as iron. BAL30072 and MC-1 are two monobactams conjugated to a dihydroxypyridone siderophore that are active against Pseudomonas aeruginosa and Acinetobacter baumannii. Here, we investigated the mechanism of action of these molecules in A. baumannii. We identified two novel TonB-dependent receptors, termed Ab-PiuA and Ab-PirA, that are required for the antimicrobial activity of both agents. Deletion of either piuA or pirA in A. baumannii resulted in 4- to 8-fold-decreased susceptibility, while their overexpression in the heterologous host P. aeruginosa increased susceptibility to the two siderophore-drug conjugates by 4- to 32-fold. The crystal structures of PiuA and PirA from A. baumannii and their orthologues from P. aeruginosa were determined. The structures revealed similar architectures; however, structural differences between PirA and PiuA point to potential differences between their cognate siderophore ligands. Spontaneous mutants, selected upon exposure to BAL30072, harbored frameshift mutations in either the ExbD3 or the TonB3 protein of A. baumannii, forming the cytoplasmic-membrane complex providing the energy for the siderophore translocation process. The results of this study provide insight for the rational design of novel siderophore-drug conjugates against problematic Gram-negative pathogens.
|
Mar 2017
|
|
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Lucile
Moynie
,
Robert
Schnell
,
Stephen A.
Mcmahon
,
Tatyana
Sandalova
,
Wassila Abdelli
Boulkerou
,
Jason W.
Schmidberger
,
Magnus
Alphey
,
Cyprian
Cukier
,
Fraser
Duthie
,
Jolanta
Kopec
,
Huanting
Liu
,
Agata
Jacewicz
,
William N.
Hunter
,
James H.
Naismith
,
Gunter
Schneider
Open Access
Abstract: Bacterial infections are increasingly difficult to treat owing to the spread of antibiotic resistance. A major concern is Gram-negative bacteria, for which the discovery of new antimicrobial drugs has been particularly scarce. In an effort to accelerate early steps in drug discovery, the EU-funded AEROPATH project aims to identify novel targets in the opportunistic pathogen Pseudomonas aeruginosa by applying a multidisciplinary approach encompassing target validation, structural characterization, assay development and hit identification from small-molecule libraries. Here, the strategies used for target selection are described and progress in protein production and structure analysis is reported. Of the 102 selected targets, 84 could be produced in soluble form and the de novo structures of 39 proteins have been determined. The crystal structures of eight of these targets, ranging from hypothetical unknown proteins to metabolic enzymes from different functional classes (PA1645, PA1648, PA2169, PA3770, PA4098, PA4485, PA4992 and PA5259), are reported here. The structural information is expected to provide a firm basis for the improvement of hit compounds identified from fragment-based and high-throughput screening campaigns.
|
Jan 2013
|
|
