I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
|
Kuang-Ting
Ko
,
Frank
Lennartz
,
David
Mekhaiel
,
Bora
Guloglu
,
Arianna
Marini
,
Danielle J.
Deuker
,
Carole A.
Long
,
Matthijs
Jore
,
Kazutoyo
Miura
,
Sumi
Biswas
,
Matthew K.
Higgins
Diamond Proposal Number(s):
[23459]
Open Access
Abstract: An effective malaria vaccine remains a global health priority and vaccine immunogens which prevent transmission of the parasite will have important roles in multi-component vaccines. One of the most promising candidates for inclusion in a transmission-blocking malaria vaccine is the gamete surface protein Pfs48/45, which is essential for development of the parasite in the mosquito midgut. Indeed, antibodies which bind Pfs48/45 can prevent transmission if ingested with the parasite as part of the mosquito bloodmeal. Here we present the structure of full-length Pfs48/45, showing its three domains to form a dynamic, planar, triangular arrangement. We reveal where transmission-blocking and non-blocking antibodies bind on Pfs48/45. Finally, we demonstrate that antibodies which bind across this molecule can be transmission-blocking. These studies will guide the development of future Pfs48/45-based vaccine immunogens.
|
Sep 2022
|
|
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Diamond Proposal Number(s):
[23459]
Abstract: Chromosome segregation requires assembly of the macromolecular kinetochore complex onto centromeric DNA. While most eukaryotes have canonical kinetochore proteins that are widely conserved among eukaryotes, evolutionarily divergent kinetoplastids have a unique set of kinetochore proteins. Little is known about the mechanism of kinetochore assembly in kinetoplastids. Here we characterize two homologous kinetoplastid kinetochore proteins, KKT2 and KKT3, that constitutively localize at centromeres. They have three domains that are highly conserved among kinetoplastids: an N-terminal kinase domain of unknown function, the centromere localization domain in the middle, and the C-terminal domain that has weak similarity to polo boxes of Polo-like kinases. We show that the kinase activity of KKT2 is essential for accurate chromosome segregation, while that of KKT3 is dispensable for cell growth in Trypanosoma brucei. Crystal structures of their divergent polo boxes reveal differences between KKT2 and KKT3. We also show that the divergent polo boxes of KKT3 are sufficient to recruit KKT2 in trypanosomes. Furthermore, we demonstrate that the divergent polo boxes of KKT2 directly interact with KKT1 and that KKT1 interacts with KKT6. These results show that the divergent polo boxes of KKT2 and KKT3 are protein-protein interaction domains, which initiate kinetochore assembly in T. brucei.
|
Sep 2022
|
|
B21-High Throughput SAXS
|
Olivia J. S.
Macleod
,
Alexander D.
Cook
,
Helena
Webb
,
Mandy
Crow
,
Roisin
Burns
,
Maria
Redpath
,
Stefanie
Seisenberger
,
Camilla E.
Trevor
,
Lori
Peacock
,
Angela
Schwede
,
Nicola
Kimblin
,
Amanda F.
Francisco
,
Julia
Pepperl
,
Steve
Rust
,
Paul
Voorheis
,
Wendy
Gibson
,
Martin C.
Taylor
,
Matthew K.
Higgins
,
Mark
Carrington
Diamond Proposal Number(s):
[23459]
Open Access
Abstract: African trypanosomes are extracellular pathogens of mammals and are exposed to the adaptive and innate immune systems. Trypanosomes evade the adaptive immune response through antigenic variation, but little is known about how they interact with components of the innate immune response, including complement. Here we demonstrate that an invariant surface glycoprotein, ISG65, is a receptor for complement component 3 (C3). We show how ISG65 binds to the thioester domain of C3b. We also show that C3 contributes to control of trypanosomes during early infection in a mouse model and provide evidence that ISG65 is involved in reducing trypanosome susceptibility to C3-mediated clearance. Deposition of C3b on pathogen surfaces, such as trypanosomes, is a central point in activation of the complement system. In ISG65, trypanosomes have evolved a C3 receptor which diminishes the downstream effects of C3 deposition on the control of infection.
|
Aug 2022
|
|
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Mark A.
Tye
,
N. Connor
Payne
,
Catrine
Johansson
,
Kritika
Singh
,
Sofia A.
Santos
,
Lọla
Fagbami
,
Akansha
Pant
,
Kayla
Sylvester
,
Madeline R.
Luth
,
Sofia
Marques
,
Malcolm
Whitman
,
Maria M.
Mota
,
Elizabeth A.
Winzeler
,
Amanda K.
Lukens
,
Emily R.
Derbyshire
,
Udo
Oppermann
,
Dyann F.
Wirth
,
Ralph
Mazitschek
Diamond Proposal Number(s):
[19301]
Open Access
Abstract: The development of next-generation antimalarials that are efficacious against the human liver and asexual blood stages is recognized as one of the world’s most pressing public health challenges. In recent years, aminoacyl-tRNA synthetases, including prolyl-tRNA synthetase, have emerged as attractive targets for malaria chemotherapy. We describe the development of a single-step biochemical assay for Plasmodium and human prolyl-tRNA synthetases that overcomes critical limitations of existing technologies and enables quantitative inhibitor profiling with high sensitivity and flexibility. Supported by this assay platform and co-crystal structures of representative inhibitor-target complexes, we develop a set of high-affinity prolyl-tRNA synthetase inhibitors, including previously elusive aminoacyl-tRNA synthetase triple-site ligands that simultaneously engage all three substrate-binding pockets. Several compounds exhibit potent dual-stage activity against Plasmodium parasites and display good cellular host selectivity. Our data inform the inhibitor requirements to overcome existing resistance mechanisms and establish a path for rational development of prolyl-tRNA synthetase-targeted anti-malarial therapies.
|
Aug 2022
|
|
|
|
Open Access
Abstract: Since its discovery, bovine theileriosis has caused major socioeconomic losses in sub-Saharan Africa. Acaricide resistance of the intermediate host, paucity of therapeutics, and lack of sufficiently cross-protective vaccines increase the risk of parasite spread due to global warming. Here, we highlight three important areas that require investigation to develop next-generation vaccines.
|
Aug 2022
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
|
Open Access
Abstract: Schistosomiasis is the most severe water-borne disease in the tropics and subtropics, infecting more than 252 million people worldwide, but is most prevalent in Sub-Saharan Africa. Reduced efficacy of Praziquantel, the gold standard of treatment against the disease, and associated reports of drug resistance have diminished confidence in the drug, prompting efforts toward developing alternative or improved forms of medication. The Universal Stress G4LZI3 protein has been identified as a potential ‘lead' molecule. This manuscript reports the expression, purification, crystallization, and preliminary crystallographic data of the protein. Results showed the best diffraction resolution at 3.05 Å and that the crystals belong to space group P212121, with four molecules within the unit cell. Processing of the data, model building, and refinement were achieved by Phenix.mr_rosetta, Coot, and other Phenix programs, respectively, with the final produced structure possessing alpha helices, beta sheets, and a 310 helix. Further research aims to improve the model and use it for structure-based drug design.
|
Aug 2022
|
|
I03-Macromolecular Crystallography
|
Rachel
Milne
,
Natalie
Wiedemar
,
Victoriano
Corpas-Lopez
,
Eoin
Moynihan
,
Richard J.
Wall
,
Alice
Dawson
,
David A.
Robinson
,
Sharon M.
Shepherd
,
Robert J.
Smith
,
Irene
Hallyburton
,
John M.
Post
,
Karen
Dowers
,
Leah S.
Torrie
,
Ian H.
Gilbert
,
Beatriz
Baragaña
,
Stephen
Patterson
,
Susan
Wyllie
Diamond Proposal Number(s):
[19844]
Open Access
Abstract: There is a pressing need for new medicines to prevent and treat malaria. Most antimalarial drug discovery is reliant upon phenotypic screening. However, with the development of improved target validation strategies, target-focused approaches are now being utilized. Here, we describe the development of a toolkit to support the therapeutic exploitation of a promising target, lysyl tRNA synthetase (PfKRS). The toolkit includes resistant mutants to probe resistance mechanisms and on-target engagement for specific chemotypes; a hybrid KRS protein capable of producing crystals suitable for ligand soaking, thus providing high-resolution structural information to guide compound optimization; chemical probes to facilitate pulldown studies aimed at revealing the full range of specifically interacting proteins and thermal proteome profiling (TPP); as well as streamlined isothermal TPP methods to provide unbiased confirmation of on-target engagement within a biologically relevant milieu. This combination of tools and methodologies acts as a template for the development of future target-enabling packages.
|
Aug 2022
|
|
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[20221]
Open Access
Abstract: The temporary or permanent chemical modification of biomolecules is a crucial aspect in the physiology of all living species. However, while some modules are well characterised also in insects, others did not receive the same attention. This holds true for sulfo-conjugation that is catalysed by cytosolic sulfotransferases (SULT), a central component of the metabolism of endogenous low molecular weight molecules and xenobiotics. In particular, limited information is available about the functional roles of the mosquito predicted enzymes annotated as SULTs in genomic databases. The herein described research is the first example of a biochemical and structural study of a SULT of a mosquito species, in general, and of the malaria vector Anopheles gambiae in particular. We confirmed that the AGAP001425 transcript displays a peculiar expression pattern that is suggestive of a possible involvement in modulating the mosquito reproductive tissues physiology, a fact that could raise attention on the enzyme as a potential target for insect-containment strategies. The crystal structures of the enzyme in alternative ligand-bound states revealed elements distinguishing AgSULT-001425 from other characterized SULTs, including a peculiar conformational plasticity of a discrete region that shields the catalytic cleft and that could play a main role in the dynamics of the reaction and in the substrate selectivity of the enzyme. Along with further in vitro biochemical studies, our structural investigations could provide a framework for the discovery of small-molecule inhibitors to assess the effect of interfering with AgSULT-001425-mediated catalysis at the organismal level.
|
Jul 2022
|
|
NONE-No attached Diamond beamline
|
Open Access
Abstract: Trypanothione reductase (TR) is a key factor in the redox homeostasis of trypanosomatid parasites, critical for survival in the hostile oxidative environment generated by the host to fight infection. TR is considered an attractive target for the development of new trypanocidal agents as it is essential for parasite survival but has no close homolog in humans. However, the high efficiency and turnover of TR challenging targets since only potent inhibitors, with nanomolar IC50, can significantly affect parasite redox state and viability. To aid the design of effective compounds targeting TR, we performed a fragment-based crystal screening at the Diamond Light Source XChem facility using a library optimized for follow-up synthesis steps. The experiment, allowing for testing over 300 compounds, resulted in the identification of 12 new ligands binding five different sites. Interestingly, the screening revealed the existence of an allosteric pocket close to the NADPH binding site, named the “doorstop pocket” since ligands binding at this site interfere with TR activity by hampering the “opening movement” needed to allow cofactor binding. The second remarkable site, known as the Z-site, identified by the screening, is located within the large trypanothione cavity but corresponds to a region not yet exploited for inhibition. The fragments binding to this site are close to each other and have some remarkable features making them ideal for follow-up optimization as a piperazine moiety in three out of five fragments.
|
Jul 2022
|
|
I03-Macromolecular Crystallography
|
Samuel L.
Freeman
,
Vera
Skafar
,
Hanna
Kwon
,
Alistair J.
Fielding
,
Peter C. E.
Moody
,
Alejandra
Martínez
,
Federico
Issoglio
,
Lucas
Inchausti
,
Pablo
Smircich
,
Ari
Zeida
,
Lucía
Piacenza
,
Rafael
Radi
,
Emma L.
Raven
Diamond Proposal Number(s):
[23269]
Open Access
Abstract: The protozoan parasite Trypanosoma cruzi is the causative agent of American trypanosomiasis, otherwise known as Chagas disease. To survive in the host, the T. cruzi parasite needs antioxidant defence systems. One of these is a hybrid heme peroxidase, the T. cruzi ascorbate peroxidase-cytochrome c peroxidase enzyme (TcAPx-CcP). TcAPx-CcP has high sequence identity to members of the class I peroxidase family, notably ascorbate peroxidase (APX) and cytochrome c peroxidase (CcP), as well as a mitochondrial peroxidase from Leishmania major (LmP). The aim of this work was to solve the structure and examine the reactivity of the TcAPx-CcP enzyme. Low temperature electron paramagnetic resonance (EPR) spectra support the formation of an exchange-coupled [Fe(IV)=O Trp233•+] Compound I radical species, analogous to that used in CcP and LmP. We demonstrate that TcAPx-CcP is similar in overall structure to APX and CcP, but there are differences in the substrate binding regions. Furthermore, the electron transfer pathway from cytochrome c to the heme in CcP and LmP is preserved in the TcAPx-CcP structure. Integration of steady state kinetic experiments, molecular dynamic simulations, and bioinformatic analyses indicates that TcAPx-CcP preferentially oxidizes cytochrome c, but is still competent for oxididation of ascorbate. The results reveal that TcAPx-CcP is a credible cytochrome c peroxidase which can also bind and use ascorbate in host cells, where concentrations are in the millimolar range. Thus, kinetically and functionally TcAPx-CcP can be considered a hybrid peroxidase.
|
Jun 2022
|
|
