I03-Macromolecular Crystallography
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Open Access
Abstract: Natural killer (NK) cells use inhibitory and activating immune receptors to differentiate between human cells and pathogens. Signalling by these receptors determines whether an NK cell becomes activated and destroys a target cell. In some cases, such as killer immunoglobulin-like receptors, immune receptors are found in pairs, with inhibitory and activating receptors containing nearly identical extracellular ligand-binding domains coupled to different intracellular signalling domains1. Previous studies showed that repetitive interspersed family (RIFIN) proteins, displayed on the surfaces of Plasmodium falciparum-infected erythrocytes, can bind to inhibitory immune receptors and dampen NK cell activation2,3, reducing parasite killing. However, no pathogen-derived ligand has been identified for any human activating receptor. Here we identified a clade of RIFINs that bind to inhibitory immune receptor KIR2DL1 more strongly than KIR2DL1 binds to the human ligand (MHC class I). This interaction mediates inhibitory signalling and suppresses the activation of KIR2DL1-expressing NK cells. We show that KIR2DL1-binding RIFINs are abundant in field-isolated strains from both Africa and Asia and reveal how the two RIFINs bind to KIR2DL1. The RIFIN binding surface of KIR2DL1 is conserved in the cognate activating immune receptor KIR2DS1. We find that KIR2DL1-binding RIFINs can also bind to KIR2DS1, resulting in the activation of KIR2DS1-expressing NK cells. This study demonstrates that activating killer immunoglobulin-like receptors can recruit NK cells to target a pathogen and reveals a potential role for activating immune receptors in controlling malaria infection.
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Jun 2025
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[30489]
Open Access
Abstract: Crystallographic fragment screening is a powerful methodology that enables the identification of low molecular weight ligands and has shown great promises in drug discovery. In this work we report the results of a fragment screening carried out in an effort to further map the cavities of trypanothione reductase from Trypanosoma brucei (TbTR), a critical target for drug design against human African trypanosomiases (HAT), for which efficient and non-toxic trypanocidal drugs are lacking. Moreover, the conservation of trypanothione reductase among trypanosomatids, including Leishmania, could facilitate the design of a wide-spectrum drug against many parasitic diseases. At the XCHEM facility (Diamond Light Sources, United Kingdom) we performed the soaking of TbTR monoclinic crystals with fragments from DSIpoised and EubOPEN DSIp libraries and we identified eight new hits binding to different cavities of TR including the trypanothione and the NADPH binding cavities. These fragments exhibited affinities ranging from submillimolar to millimolar, as determined by surface plasmon resonance (SPR). While the newly identified fragments did not significantly alter TbTR’s enzymatic activity—consistent with the nature of low-affinity ligands—they provide valuable insights into key interactions of fragments with TR and, together with prior fragment screening campaigns, pave the way towards follow-up chemical optimization into lead compounds.
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May 2025
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I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: Plasmodium falciparum plasmepsin X (PMX) has become a target of choice for the development of new antimalarial drugs due to its essential role across the parasite life cycle. Here we describe the 1.7Å crystallographic structure of PMX noncovalently bound to a potent macrocyclic peptidomimetic inhibitor (7k) possessing a hydroxyethylamine (HEA) scaffold. Upon 7k binding, the enzyme adopts a novel conformation, with significant involvement of the S2’S2 loop (M526-H536) and the S2 flap (F311-G314). This results in partial closure of the active site with widespread interactions in both the prime (S’) and the non-prime (S) sites of PMX. The catalytic aspartate residues D266 and D467 directly interact with the HEA pharmacophore. Docking of a 7k derivative, compound 7a, highlights a region in the S3 pocket near the S3 flexible loop (H242-F248) that may be key for ligand stabilisation. The dynamic nature of PMX and its propensity to undergo distinct types of induced fit upon inhibitor binding enables generation of potent inhibitors that target this essential malarial aspartic protease.
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Mar 2025
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[33825]
Abstract: Septins are cytoskeletal proteins and their interaction with membranes is crucial for their role in various cellular processes. Septins have polybasic regions (PB1 and PB2) which are important for lipid interaction. Earlier, we and others have highlighted the role of the septin C-terminal domain (CTD) to membrane interaction. However, detailed information on residues/group of residues important for such feature is lacking. In this study, we investigate the lipid-binding profile of Schistosoma mansoni Septin10 (SmSEPT10) using PIP strip and Langmuir monolayer adsorption assays. Our findings highlight the CTD as the primary domain responsible for lipid interaction in SmSEPT10, showing binding to phosphatidylinositol phosphates. SmSEPT10 CTD contains a conserved polybasic region (PB3) present in both animals and fungi septins, and a Lys (K367) within its putative amphipathic helix (AH) that we demonstrate as important for lipid binding. PB3 deletion or mutation of this Lys (K367A) strongly impairs lipid interaction. Remarkably, we observe that the AH within a construct lacking the final 43 amino acid residues is insufficient for lipid binding. Furthermore, we investigate the homocomplex formed by SmSEPT10 CTD in solution by cross-linking experiments, CD spectroscopy, SEC-MALS and SEC-SAXS. Taken together, our studies define the lipid-binding region in SmSEPT10 and offer insights into the molecular basis of septin-membrane binding. This information is particularly relevant for less-studied non-human septins, such as SmSEPT10.
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Oct 2024
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I03-Macromolecular Crystallography
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Nicola
Caldwell
,
Caroline
Peet
,
Peter
Miller
,
Beatrice L.
Colon
,
Malcolm G.
Taylor
,
Mattia
Cocco
,
Alice
Dawson
,
Iva
Lukac
,
Jose E.
Teixeira
,
Lee
Robinson
,
Laura
Frame
,
Simona
Seizova
,
Sebastian
Damerow
,
Fabio
Tamaki
,
John
Post
,
Jennifer
Riley
,
Nicole
Mutter
,
Jack C.
Hanna
,
Liam
Ferguson
,
Xiao
Hu
,
Michele
Tinti
,
Barbara
Forte
,
Neil R.
Norcross
,
Peter S.
Campbell
,
Nina
Svensen
,
Flora C.
Caldwell
,
Chimed
Jansen
,
Vincent
Postis
,
Kevin D.
Read
,
Christopher D.
Huston
,
Ian H.
Gilbert
,
Beatriz
Baragaña
,
Mattie C.
Pawlowic
Diamond Proposal Number(s):
[19844]
Abstract: Cryptosporidiosis is a diarrheal disease caused by infection with Cryptosporidium spp. parasites and is a leading cause of death in malnourished children worldwide. The only approved treatment, nitazoxanide, has limited efficacy in this at-risk patient population. Additional safe therapeutics are urgently required to tackle this unmet medical need. However, the development of anti-cryptosporidial drugs is hindered by a lack of understanding of the optimal compound properties required to treat this gastrointestinal infection. To address this knowledge gap, a diverse set of potent lysyl-tRNA synthetase inhibitors was profiled to identify optimal physicochemical and pharmacokinetic properties required for efficacy in a chronic mouse model of infection. The results from this comprehensive study illustrated the importance of balancing solubility and permeability to achieve efficacy in vivo. Our results establish in vitro criteria for solubility and permeability that are predictive of compound efficacy in vivo to guide the optimization of anti-cryptosporidial drugs. Two compounds from chemically distinct series (DDD489 and DDD508) were identified as demonstrating superior efficacy and prioritized for further evaluation. Both compounds achieved marked parasite reduction in immunocompromised mouse models and a disease-relevant calf model of infection. On the basis of these promising data, these compounds have been selected for progression to preclinical safety studies, expanding the portfolio of potential treatments for this neglected infectious disease.
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Oct 2024
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[23459]
Open Access
Abstract: There is an urgent need for improved malaria vaccine immunogens. Invasion of erythrocytes by Plasmodium falciparum is essential for its life cycle, preceding symptoms of disease and parasite transmission. Antibodies which target PfRH5 are highly effective at preventing erythrocyte invasion and the most potent growth-inhibitory antibodies bind a single epitope. Here we use structure-guided approaches to design a small synthetic immunogen, RH5-34EM which recapitulates this epitope. Structural biology and biophysics demonstrate that RH5-34EM is correctly folded and binds neutralising monoclonal antibodies with nanomolar affinity. In immunised rats, RH5-34EM induces PfRH5-targeting antibodies that inhibit parasite growth. While PfRH5-specific antibodies were induced at a lower concentration by RH5-34EM than by PfRH5, RH5-34EM induced antibodies that were a thousand-fold more growth-inhibitory as a factor of PfRH5-specific antibody concentration. Finally, we show that priming with RH5-34EM and boosting with PfRH5 achieves the best balance between antibody quality and quantity and induces the most effective growth-inhibitory response. This rationally designed vaccine immunogen is now available for use as part of future malaria vaccines, alone or in combination with other immunogens.
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Sep 2024
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I02-Macromolecular Crystallography
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Michal
Banasik
,
Valeria
Napolitano
,
Artur
Blat
,
Karim
Abdulkarim
,
Jacek
Plewka
,
Cezary
Czaplewski
,
Artur
Gieldon
,
Maciej
Kozak
,
Benedykt
Wladyka
,
Grzegorz
Popowicz
,
Grzegorz
Dubin
Abstract: Peroxisomal protein import has been identified as a valid target in trypanosomiases, an important health threat in Central and South America. The importomer is built of multiple peroxins (Pex) and structural characterization of these proteins facilitates rational inhibitor development. We report crystal structures of the Trypanosoma brucei and T. cruzi tetratricopeptide repeat domain (TPR) of the cytoplasmic peroxisomal targeting signal 1 (PTS1) receptor Pex5. The structure of the TPR domain of TbPex5 represents an apo-form of the receptor which, together with the previously determined structure of the complex of TbPex5 TPR and PTS1 demonstrate significant receptor dynamics associated with signal peptide recognition. The structure of the complex of TPR domain of TcPex5 with PTS1 provided in this study details the molecular interactions that guide signal peptide recognition at the atomic level in the pathogenic species currently perceived as the most relevant among Trypanosoma. Small – angle X – ray scattering (SAXS) data obtained in solution supports the crystallographic findings on the compaction of the TPR domains of TbPex5 and TcPex5 upon interaction with the cargo.
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Sep 2024
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Antoni R.
Blaazer
,
Abhimanyu K.
Singh
,
Lorena
Zara
,
Pierre
Boronat
,
Lady J.
Bautista
,
Steve
Irving
,
Maciej
Majewski
,
Xavier
Barril
,
Maikel
Wijtmans
,
U. Helena
Danielson
,
Geert Jan
Sterk
,
Rob
Leurs
,
Jacqueline E.
Van Muijlwijk-Koezen
,
David G.
Brown
,
Iwan
De Esch
Open Access
Abstract: In search of new opportunities to develop Trypanosoma brucei phosphodiesterase B1 (TbrPDEB1) inhibitors that have selectivity over the off-target human PDE4 (hPDE4), different stages of a fragment-growing campaign were studied using a variety of biochemical, structural, thermodynamic, and kinetic binding assays. Remarkable differences in binding kinetics were identified and this kinetic selectivity was explored with computational methods, including molecular dynamics and interaction fingerprint analyses. These studies indicate that a key hydrogen bond between GlnQ.50 and the inhibitors is exposed to a water channel in TbrPDEB1, leading to fast unbinding. This water channel is not present in hPDE4, leading to inhibitors with a longer residence time. The computer-aided drug design protocols were applied to a recently disclosed TbrPDEB1 inhibitor with a different scaffold and our results confirm that shielding this key hydrogen bond through disruption of the water channel represents a viable design strategy to develop more selective inhibitors of TbrPDEB1. Our work shows how computational protocols can be used to understand the contribution of solvent dynamics to inhibitor binding, and our results can be applied in the design of selective inhibitors for homologous PDEs found in related parasites.
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Aug 2024
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Lawrence T.
Wang
,
Andrew J. R.
Cooper
,
Brendan
Farrell
,
Kazutoyo
Miura
,
Ababacar
Diouf
,
Nicole
Müller-Sienerth
,
Cécile
Crosnier
,
Lauren
Purser
,
Payton J.
Kirtley
,
Maciej
Maciuszek
,
Jordan R.
Barrett
,
Kirsty
Mchugh
,
Rodney
Ogwang
,
Courtney
Tucker
,
Shanping
Li
,
Safiatou
Doumbo
,
Didier
Doumtabe
,
Chul-Woo
Pyo
,
Jeff
Skinner
,
Carolyn M.
Nielsen
,
Sarah E.
Silk
,
Kassoum
Kayentao
,
Aissata
Ongoiba
,
Ming
Zhao
,
Doan C.
Nguyen
,
F. Eun-Hyung
Lee
,
Angela M.
Minassian
,
Daniel E.
Geraghty
,
Boubacar
Traore
,
Robert A.
Seder
,
Brandon K.
Wilder
,
Peter D.
Crompton
,
Gavin J.
Wright
,
Carole A.
Long
,
Simon J.
Draper
,
Matthew K.
Higgins
,
Joshua
Tan
Open Access
Abstract: Plasmodium falciparum reticulocyte-binding protein homolog 5 (RH5) is the most advanced blood-stage malaria vaccine candidate and is being evaluated for efficacy in endemic regions, emphasizing the need to study the underlying antibody response to RH5 during natural infection, which could augment or counteract responses to vaccination. Here, we found that RH5-reactive B cells were rare, and circulating immunoglobulin G (IgG) responses to RH5 were short-lived in malaria-exposed Malian individuals, despite repeated infections over multiple years. RH5-specific monoclonal antibodies isolated from eight malaria-exposed individuals mostly targeted non-neutralizing epitopes, in contrast to antibodies isolated from five RH5-vaccinated, malaria-naive UK individuals. However, MAD8–151 and MAD8–502, isolated from two malaria-exposed Malian individuals, were among the most potent neutralizers out of 186 antibodies from both cohorts and targeted the same epitopes as the most potent vaccine-induced antibodies. These results suggest that natural malaria infection may boost RH5-vaccine-induced responses and provide a clear strategy for the development of next-generation RH5 vaccines.
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Jul 2024
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Jordan R.
Barrett
,
Dimitra
Pipini
,
Nathan D.
Wright
,
Andrew J. R.
Cooper
,
Giacomo
Gorini
,
Doris
Quinkert
,
Amelia M.
Lias
,
Hannah
Davies
,
Cassandra A.
Rigby
,
Maya
Aleshnick
,
Barnabas G.
Williams
,
William J.
Bradshaw
,
Neil G.
Paterson
,
Thomas
Martinson
,
Payton
Kirtley
,
Luc
Picard
,
Christine D.
Wiggins
,
Francesca R.
Donnellan
,
Lloyd D. W.
King
,
Lawrence T.
Wang
,
Jonathan F.
Popplewell
,
Sarah E.
Silk
,
Jed
De Ruiter Swain
,
Katherine
Skinner
,
Vinayaka
Kotraiah
,
Amy R.
Noe
,
Randall S.
Macgill
,
C. Richter
King
,
Ashley J.
Birkett
,
Lorraine A.
Soisson
,
Angela M.
Minassian
,
Douglas A.
Lauffenburger
,
Kazutoyo
Miura
,
Carole A.
Long
,
Brandon K.
Wilder
,
Lizbe
Koekemoer
,
Joshua
Tan
,
Carolyn M.
Nielsen
,
Kirsty
Mchugh
,
Simon J.
Draper
Diamond Proposal Number(s):
[28172]
Open Access
Abstract: The highly conserved and essential Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) has emerged as the leading target for vaccines against the disease-causing blood stage of malaria. However, the features of the human vaccine-induced antibody response that confer highly potent inhibition of malaria parasite invasion into red blood cells are not well defined. Here, we characterize 236 human IgG monoclonal antibodies, derived from 15 donors, induced by the most advanced PfRH5 vaccine. We define the antigenic landscape of this molecule and establish that epitope specificity, antibody association rate, and intra-PfRH5 antibody interactions are key determinants of functional anti-parasitic potency. In addition, we identify a germline IgG gene combination that results in an exceptionally potent class of antibody and demonstrate its prophylactic potential to protect against P. falciparum parasite challenge in vivo. This comprehensive dataset provides a framework to guide rational design of next-generation vaccines and prophylactic antibodies to protect against blood-stage malaria.
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Jul 2024
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