I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[18069]
Open Access
Abstract: The kinetochore is the macromolecular protein complex that assembles onto centromeric DNA and binds spindle microtubules. Evolutionarily divergent kinetoplastids have an unconventional set of kinetochore proteins. It remains unknown how kinetochores assemble at centromeres in these organisms. Here, we characterize KKT2 and KKT3 in the kinetoplastid parasite Trypanosoma brucei. In addition to the N-terminal kinase domain and C-terminal divergent polo boxes, these proteins have a central domain of unknown function. We show that KKT2 and KKT3 are important for the localization of several kinetochore proteins and that their central domains are sufficient for centromere localization. Crystal structures of the KKT2 central domain from two divergent kinetoplastids reveal a unique zinc-binding domain (termed the CL domain for centromere localization), which promotes its kinetochore localization in T. brucei. Mutations in the equivalent domain in KKT3 abolish its kinetochore localization and function. Our work shows that the unique central domains play a critical role in mediating the centromere localization of KKT2 and KKT3.
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Aug 2021
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19946]
Abstract: Plasmodium falciparum lysyl tRNA synthetase ( Pf KRS) represent a promising therapeutic anti‐malarial target. Cladosporin, a tool compound was identified as a selective and potent PfKRS inhibitor but lacks metabolic stability. Here, we report chemical synthesis, biological evaluation and structural characterization of analogues where the tetrahydropyran (THP) frame of cladosporin is replaced with the piperidine ring bearing functional group variations. Thermal binding, enzymatic, kinetic and parasitic assays complemented with x‐ray crystallography reveals compounds to be of moderate potency. Co‐crystals of Cla‐B and Cla‐C with PfKRS reveals key atomic configurations that allow drug binding to, and inhibition of the enzyme. Collectively, these piperidine ring scaffold inhibitors lays a framework for further structural editing and functional modifications of cladosporin scaffold to obtain a potent lead.
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May 2021
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I24-Microfocus Macromolecular Crystallography
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Stefano
De Benedetti
,
Flavio
Di Pisa
,
Enrico Mario Alessandro
Fassi
,
Marina
Cretich
,
Angelo
Musicò
,
Roberto
Frigerio
,
Alessandro
Mussida
,
Mauro
Bombaci
,
Renata
Grifantini
,
Giorgio
Colombo
,
Martino
Bolognesi
,
Romualdo
Grande
,
Nadia
Zanchetta
,
Maria Rita
Gismondo
,
Davide
Mileto
,
Alessandro
Mancon
,
Louise Jane
Gourlay
Diamond Proposal Number(s):
[5912]
Open Access
Abstract: The human parasitic disease Schistosomiasis is caused by the Schistosoma trematode flatworm that infects freshwaters in tropical regions of the world, particularly in Sub-Saharan Africa, South America, and the Far-East. It has also been observed as an emerging disease in Europe, due to increased immigration. In addition to improved therapeutic strategies, it is imperative to develop novel, rapid, and sensitive diagnostic tests that can detect the Schistosoma parasite, allowing timely treatment. Present diagnosis is difficult and involves microscopy-based detection of Schistosoma eggs in the feces. In this context, we present the 3.22 Å resolution crystal structure of the circulating antigen Serine protease inhibitor from S. mansoni (SmSPI), and we describe it as a potential serodiagnostic marker. Moreover, we identify three potential immunoreactive epitopes using in silico-based epitope mapping methods. Here, we confirm effective immune sera reactivity of the recombinant antigen, suggesting the further investigation of the protein and/or its predicted epitopes as serodiagnostic Schistosomiasis biomarkers.
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Apr 2021
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I04-Macromolecular Crystallography
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Abstract: Here we have described a systematic structure activity relationship (SAR) of a set of compounds inspired from cladosporin, a tool compound that targets parasite (Plasmodium falciparum) lysyl tRNA synthetase (KRS). Four sets of analogues, synthesized based on point changes in the chemical scaffold of cladosporin and other logical modifications and hybridizations, were assessed using high throughput enzymatic and parasitic assays along with in vitro pharmacokinetics. Co-crystallization of the most potent compound in our series (CL-2) with PfKRS revealed its structural basis of enzymatic binding and potency. Further, we report that CL-2 has performed better than cladosporin in terms of metabolic stability. It thus represents a new lead for further optimization toward the development of antimalarial drugs. Collectively, along with a lead compound, the series offers insights on how even the slightest chemical modification might play an important role in enhancing or decreasing the potency of a chemical scaffold.
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Apr 2021
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[23459]
Open Access
Abstract: The kinetochore is the macromolecular machinery that drives chromosome segregation by interacting with spindle microtubules. Kinetoplastids (such as Trypanosoma brucei), a group of evolutionarily divergent eukaryotes, have a unique set of kinetochore proteins that lack any significant homology to canonical kinetochore components. To date, KKT4 is the only kinetoplastid kinetochore protein that is known to bind microtubules. Here we use X-ray crystallography, NMR spectroscopy, and crosslinking mass spectrometry to characterize the structure and dynamics of KKT4. We show that its microtubule-binding domain consists of a coiled-coil structure followed by a positively charged disordered tail. The structure of the C-terminal BRCT domain of KKT4 reveals that it is likely a phosphorylation-dependent protein-protein interaction domain. The BRCT domain interacts with the N-terminal region of the KKT4 microtubule-binding domain and with a phosphopeptide derived from KKT8. Taken together, these results provide structural insights into the unconventional kinetoplastid kinetochore protein KKT4.
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Apr 2021
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Nur Zazarina
Ramly
,
Samuel R.
Dix
,
Sergey N.
Ruzheinikov
,
Svetlana E.
Sedelnikova
,
Patrick J.
Baker
,
Yock-Ping
Chow
,
Fiona M.
Tomley
,
Damer P.
Blake
,
Kiew-Lian
Wan
,
Sheila
Nathan
,
David W.
Rice
Diamond Proposal Number(s):
[1218, 24447]
Open Access
Abstract: In infections by apicomplexan parasites including Plasmodium, Toxoplasma gondii, and Eimeria, host interactions are mediated by proteins including families of membrane-anchored cysteine-rich surface antigens (SAGs) and SAG-related sequences (SRS). Eimeria tenella causes caecal coccidiosis in chickens and has a SAG family with over 80 members making up 1% of the proteome. We have solved the structure of a representative E. tenella SAG, EtSAG19, revealing that, despite a low level of sequence similarity, the entire Eimeria SAG family is unified by its three-layer αβα fold which is related to that of the CAP superfamily. Furthermore, sequence comparisons show that the Eimeria SAG fold is conserved in surface antigens of the human coccidial parasite Cyclospora cayetanensis but this fold is unrelated to that of the SAGs/SRS proteins expressed in other apicomplexans including Plasmodium species and the cyst-forming coccidia Toxoplasma gondii, Neospora caninum and Besnoitia besnoiti. However, despite having very different structures, Consurf analysis showed that Eimeria SAG and Toxoplasma SRS families each exhibit marked hotspots of sequence hypervariability that map to their surfaces distal to the membrane anchor. This suggests that the primary and convergent purpose of the different structures is to provide a platform onto which sequence variability can be imposed.
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Mar 2021
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Krios I-Titan Krios I at Diamond
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Open Access
Abstract: The biogenic formation of hemozoin crystals, a crucial process in heme detoxification by the malaria parasite, is reviewed as an antimalarial drug target. We first focus on the in‐vivo formation of hemozoin. A model is presented, based on native‐contrast 3D imaging obtained by X‐ray and electron microscopy, that hemozoin nucleates at the inner membrane leaflet of the parasitic digestive vacuole, and grows in the adjacent aqueous medium. Having observed quantities of hemoglobin and hemozoin in the digestive vacuole, we present a model that heme liberation from hemoglobin and hemozoin formation is an assembly‐line process. The crystallization is preceded by reaction between heme monomers yielding hematin dimers involving fewer types of isomers than in synthetic hemozoin; this is indicative of protein‐induced dimerization. Models of antimalarial drugs binding onto hemozoin surfaces are reviewed. This is followed by a description of bromoquine, a chloroquine drug analogue, capping a significant fraction of hemozoin surfaces within the digestive vacuole and accumulation of the drug, presumably a bromoquine–hematin complex, at the vacuole's membrane.
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Mar 2021
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Iain W.
Mcnae
,
James
Kinkead
,
Divya
Malik
,
Li-Hsuan
Yen
,
Martin K.
Walker
,
Chris
Swain
,
Scott P.
Webster
,
Nick
Gray
,
Peter M.
Fernandes
,
Elmarie
Myburgh
,
Elizabeth
Blackburn
,
Ryan
Ritchie
,
Carol
Austin
,
Martin A.
Wear
,
Adrian J.
Highton
,
Andrew J.
Keats
,
Antonio
Vong
,
Jacqueline
Dornan
,
Jeremy C.
Mottram
,
Paul A. M.
Michels
,
Simon
Pettit
,
Malcolm D.
Walkinshaw
Diamond Proposal Number(s):
[9487, 13550]
Open Access
Abstract: The parasitic protist Trypanosoma brucei is the causative agent of Human African Trypanosomiasis, also known as sleeping sickness. The parasite enters the blood via the bite of the tsetse fly where it is wholly reliant on glycolysis for the production of ATP. Glycolytic enzymes have been regarded as challenging drug targets because of their highly conserved active sites and phosphorylated substrates. We describe the development of novel small molecule allosteric inhibitors of trypanosome phosphofructokinase (PFK) that block the glycolytic pathway resulting in very fast parasite kill times with no inhibition of human PFKs. The compounds cross the blood brain barrier and single day oral dosing cures parasitaemia in a stage 1 animal model of human African trypanosomiasis. This study demonstrates that it is possible to target glycolysis and additionally shows how differences in allosteric mechanisms may allow the development of species-specific inhibitors to tackle a range of proliferative or infectious diseases.
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Feb 2021
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I03-Macromolecular Crystallography
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Johan
Zeelen
,
Monique
Van Straaten
,
Joseph
Verdi
,
Alexander
Hempelmann
,
Hamidreza
Hashemi
,
Kathryn
Perez
,
Philip D.
Jeffrey
,
Silvan
Hälg
,
Natalie
Wiedemar
,
Pascal
Mäser
,
F. Nina
Papavasiliou
,
C. Eric
Stebbins
Diamond Proposal Number(s):
[18989]
Abstract: Suramin has been a primary early-stage treatment for African trypanosomiasis for nearly 100 yr. Recent studies revealed that trypanosome strains that express the variant surface glycoprotein (VSG) VSGsur possess heightened resistance to suramin. Here, we show that VSGsur binds tightly to suramin but other VSGs do not. By solving high-resolution crystal structures of VSGsur and VSG13, we also demonstrate that these VSGs define a structurally divergent subgroup of the coat proteins. The co-crystal structure of VSGsur with suramin reveals that the chemically symmetric drug binds within a large cavity in the VSG homodimer asymmetrically, primarily through contacts of its central benzene rings. Structure-based, loss-of-contact mutations in VSGsur significantly decrease the affinity to suramin and lead to a loss of the resistance phenotype. Altogether, these data show that the resistance phenotype is dependent on the binding of suramin to VSGsur, establishing that the VSG proteins can possess functionality beyond their role in antigenic variation.
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Jan 2021
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I24-Microfocus Macromolecular Crystallography
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Manmohan
Sharma
,
Nipun
Malhotra
,
Manickam
Yogavel
,
Karl
Harlos
,
Bruno
Melillo
,
Eamon
Comer
,
Arthur
Gonse
,
Suhel
Parvez
,
Branko
Mitasev
,
Francis G.
Fang
,
Stuart L.
Schreiber
,
Amit
Sharma
Diamond Proposal Number(s):
[19946]
Open Access
Abstract: The inhibition of Plasmodium cytosolic phenylalanine tRNA-synthetase (cFRS) by a novel series of bicyclic azetidines has shown the potential to prevent malaria transmission, provide prophylaxis, and offer single-dose cure in animal models of malaria. To date, however, the molecular basis of Plasmodium cFRS inhibition by bicyclic azetidines has remained unknown. Here, we present structural and biochemical evidence that bicyclic azetidines are competitive inhibitors of L-Phe, one of three substrates required for the cFRS-catalyzed aminoacylation reaction that underpins protein synthesis in the parasite. Critically, our co-crystal structure of a PvcFRS-BRD1389 complex shows that the bicyclic azetidine ligand binds to two distinct sub-sites within the PvcFRS catalytic site. The ligand occupies the L-Phe site along with an auxiliary cavity and traverses past the ATP binding site. Given that BRD1389 recognition residues are conserved amongst apicomplexan FRSs, this work lays a structural framework for the development of drugs against both Plasmodium and related apicomplexans.
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Jan 2021
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