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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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Abstract: Mycobacterium tuberculosis is responsible for more than 1.6 million deaths each year. One potential antibacterial target in M. tuberculosis is filamentous temperature sensitive protein Z (FtsZ), which is the bacterial homologue of mammalian tubulin, a validated cancer target. M. tuberculosis FtsZ function is essential, with its inhibition leading to arrest of cell division, elongation of the bacterial cell and eventual cell death. However, the development of potent inhibitors against FtsZ has been a challenge owing to the lack of structural information. Here we report multiple crystal structures of M. tuberculosis FtsZ in complex with a coumarin analogue. The 4-hydroxycoumarin binds exclusively to two novel cryptic pockets in nucleotide-free FtsZ, but not to the binary FtsZ-GTP or GDP complexes. Our findings provide a detailed understanding of the molecular basis for cryptic pocket formation, controlled by the conformational flexibility of the H7 helix, and thus reveal an important structural and mechanistic rationale for coumarin antibacterial activity.
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Jul 2021
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[19946]
Open Access
Abstract: Influenza A viruses of the H1N1 and H3N2 subtypes are responsible for seasonal epidemic events. The influenza nucleoprotein (NP) binds to the viral genomic RNA and is essential for its replication. Efforts are under way to produce therapeutics and vaccines targeting the NP. Despite this, no structure of an NP from an H3N2 virus has previously been determined. Here, the structure of the A/Northern Territory/60/1968 (H3N2) influenza virus NP is presented at 2.2 Å resolution. The structure is highly similar to those of the A/WSN/1933 (H1N1) and A/Hong Kong/483/97 (H5N1) NPs. Nonconserved amino acids are widely dispersed both at the sequence and structural levels. A movement of the 73–90 RNA-binding loop is observed to be the key difference between the structure determined here and previous structures. The data presented here increase the understanding of structural conservation amongst influenza NPs and may aid in the design of universal interventions against influenza.
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Jul 2021
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I24-Microfocus Macromolecular Crystallography
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Agata
Butryn
,
Philipp S.
Simon
,
Pierre
Aller
,
Philip
Hinchliffe
,
Ramzi N.
Massad
,
Gabriel
Leen
,
Catherine L.
Tooke
,
Isabel
Bogacz
,
In-Sik
Kim
,
Asmit
Bhowmick
,
Aaron S.
Brewster
,
Nicholas E.
Devenish
,
Jurgen
Brem
,
Jos J. A. G.
Kamps
,
Pauline A.
Lang
,
Patrick
Rabe
,
Danny
Axford
,
John H.
Beale
,
Bradley
Davy
,
Ali
Ebrahim
,
Julien
Orlans
,
Selina L. S.
Storm
,
Tiankun
Zhou
,
Shigeki
Owada
,
Rie
Tanaka
,
Kensuke
Tono
,
Gwyndaf
Evans
,
Robin L.
Owen
,
Frances A.
Houle
,
Nicholas K.
Sauter
,
Christopher J.
Schofield
,
James
Spencer
,
Vittal K.
Yachandra
,
Junko
Yano
,
Jan F.
Kern
,
Allen M.
Orville
Diamond Proposal Number(s):
[19458, 25260]
Open Access
Abstract: Serial femtosecond crystallography has opened up many new opportunities in structural biology. In recent years, several approaches employing light-inducible systems have emerged to enable time-resolved experiments that reveal protein dynamics at high atomic and temporal resolutions. However, very few enzymes are light-dependent, whereas macromolecules requiring ligand diffusion into an active site are ubiquitous. In this work we present a drop-on-drop sample delivery system that enables the study of enzyme-catalyzed reactions in microcrystal slurries. The system delivers ligand solutions in bursts of multiple picoliter-sized drops on top of a larger crystal-containing drop inducing turbulent mixing and transports the mixture to the X-ray interaction region with temporal resolution. We demonstrate mixing using fluorescent dyes, numerical simulations and time-resolved serial femtosecond crystallography, which show rapid ligand diffusion through microdroplets. The drop-on-drop method has the potential to be widely applicable to serial crystallography studies, particularly of enzyme reactions with small molecule substrates.
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Jul 2021
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[23364]
Open Access
Abstract: Lipoproteins serve diverse functions in the bacterial cell and some are essential for survival. Some lipoproteins are adjuvants eliciting responses from the innate immune system of the host. The growing list of membrane enzymes responsible for lipoprotein synthesis includes the recently discovered lipoprotein intramolecular transacylase, Lit. Lit creates a lipoprotein that is less immunogenic, possibly enabling the bacteria to gain a foothold in the host by stealth. Here, we report the crystal structure of the Lit enzyme from Bacillus cereus and describe its mechanism of action. Lit consists of four transmembrane helices with an extracellular cap. Conserved residues map to the cap-membrane interface. They include two catalytic histidines that function to effect unimolecular transacylation. The reaction involves acyl transfer from the sn-2 position of the glyceryl moiety to the amino group on the N-terminal cysteine of the substrate via an 8-membered ring intermediate. Transacylation takes place in a confined aromatic residue-rich environment that likely evolved to bring distant moieties on the substrate into proximity and proper orientation for catalysis.
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Jul 2021
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I24-Microfocus Macromolecular Crystallography
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Paul
White
,
Samuel F.
Haysom
,
Matthew G.
Iadanza
,
Anna J.
Higgins
,
Jonathan M.
Machin
,
James M.
Whitehouse
,
Jim E.
Horne
,
Bob
Schiffrin
,
Charlotte
Carpenter-Platt
,
Antonio N.
Calabrese
,
Kelly M.
Storek
,
Steven T.
Rutherford
,
David J.
Brockwell
,
Neil A.
Ranson
,
Sheena E.
Radford
Diamond Proposal Number(s):
[19248]
Open Access
Abstract: The folding of β-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the β-barrel assembly machinery (BAM). How lateral opening in the β-barrel of the major subunit BamA assists in OMP folding, and the contribution of membrane disruption to BAM catalysis remain unresolved. Here, we use an anti-BamA monoclonal antibody fragment (Fab1) and two disulphide-crosslinked BAM variants (lid-locked (LL), and POTRA-5-locked (P5L)) to dissect these roles. Despite being lethal in vivo, we show that all complexes catalyse folding in vitro, albeit less efficiently than wild-type BAM. CryoEM reveals that while Fab1 and BAM-P5L trap an open-barrel state, BAM-LL contains a mixture of closed and contorted, partially-open structures. Finally, all three complexes globally destabilise the lipid bilayer, while BamA does not, revealing that the BAM lipoproteins are required for this function. Together the results provide insights into the role of BAM structure and lipid dynamics in OMP folding.
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Jul 2021
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Aleksandra
Szykowska
,
Yu
Chen
,
Thomas B.
Smith
,
Charlotta
Preger
,
Jingjie
Yang
,
Dongming
Qian
,
Shubhashish M.
Mukhopadhyay
,
Edvard
Wigren
,
Stephen J.
Neame
,
Susanne
Gräslund
,
Helena
Persson
,
Peter J.
Atkinson
,
Elena
Di Daniel
,
Emma
Mead
,
John
Wang
,
John B.
Davis
,
Nicola A.
Burgess-Brown
,
Alex N.
Bullock
Diamond Proposal Number(s):
[15433]
Open Access
Abstract: Mutations in TREM2, a receptor expressed by microglia in the brain, are associated with an increased risk of neurodegeneration, including Alzheimer's disease. Numerous studies support a role for TREM2 in sensing damaging stimuli and triggering signaling cascades necessary for neuroprotection. Despite its significant role, ligands and regulators of TREM2 activation, and the mechanisms governing TREM2-dependent responses and its cleavage from the membrane, remain poorly characterized. Here, we present phage display generated antibody single-chain variable fragments (scFvs) to human TREM2 immunoglobulin-like domain. Co-crystal structures revealed the binding of two scFvs to an epitope on the TREM2 domain distal to the putative ligand-binding site. Enhanced functional activity was observed for oligomeric scFv species, which inhibited the production of soluble TREM2 in a HEK293 cell model. We hope that detailed characterization of their epitopes and properties will facilitate the use of these renewable binders as structural and functional biology tools for TREM2 research.
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Jul 2021
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I24-Microfocus Macromolecular Crystallography
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Gabriele
Cerutti
,
Elena
Gugole
,
Linda Celeste
Montemiglio
,
Annick
Turbé-Doan
,
Dehbia
Chena
,
David
Navarro
,
Anne
Lomascolo
,
François
Piumi
,
Cécile
Exertier
,
Ida
Freda
,
Beatrice
Vallone
,
Eric
Record
,
Carmelinda
Savino
,
Giuliano
Sciara
Open Access
Abstract: Background: Fungal glucose dehydrogenases (GDHs) are FAD-dependent enzymes belonging to the glucose-methanol-choline oxidoreductase superfamily. These enzymes are classified in the “Auxiliary Activity” family 3 (AA3) of the Carbohydrate-Active enZymes database, and more specifically in subfamily AA3_2, that also includes the closely related flavoenzymes aryl-alcohol oxidase and glucose 1-oxidase. Based on sequence similarity to known fungal GDHs, an AA3_2 enzyme active on glucose was identified in the genome of Pycnoporus cinnabarinus, a model Basidiomycete able to completely degrade lignin. Results: In our work, substrate screening and functional characterization showed an unexpected preferential activity of this enzyme toward oligosaccharides containing a β(1→3) glycosidic bond, with the highest efficiency observed for the disaccharide laminaribiose. Despite its sequence similarity to GDHs, we defined a novel enzymatic activity, namely oligosaccharide dehydrogenase (ODH), for this enzyme. The crystallographic structures of ODH in the sugar-free form and in complex with glucose and laminaribiose unveiled a peculiar saccharide recognition mechanism which is not shared with previously characterized AA3 oxidoreductases and accounts for ODH preferential activity toward oligosaccharides. The sugar molecules in the active site of ODH are mainly stabilized through CH-π interactions with aromatic residues rather than through hydrogen bonds with highly conserved residues, as observed instead for the fungal glucose dehydrogenases and oxidases characterized to date. Finally, three sugar-binding sites were identified on ODH external surface, which were not previously observed and might be of importance in the physiological scenario. Conclusions: Structure–function analysis of ODH is consistent with its role as an auxiliary enzyme in lignocellulose degradation and unveils yet another enzymatic function within the AA3 family of the Carbohydrate-Active enZymes database. Our findings allow deciphering the molecular determinants of substrate binding and provide insight into the physiological role of ODH, opening new perspectives to exploit biodiversity for lignocellulose transformation into fuels and chemicals.
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Jul 2021
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[20113]
Open Access
Abstract: N-glycosylation is one of the most abundant post-translational modifications of proteins, essential for many physiological processes, including protein folding, protein stability, oligomerization and aggregation, and molecular recognition events. Defects in the N-glycosylation pathway cause diseases that are classified as congenital disorders of glycosylation. The ability to manipulate protein N-glycosylation is critical not only to our fundamental understanding of biology, but also for the development of new drugs for a wide range of human diseases. Chemoenzymatic synthesis using engineered endo-β-N-acetylglucosaminidases (ENGases) has been used extensively to modulate the chemistry of N-glycosylated proteins. However, defining the molecular mechanisms by which ENGases specifically recognize and process N-glycans remains a major challenge. Here we present the X-ray crystal structure of the ENGase EndoBT-3987 from Bacteroides thetaiotaomicron in complex with a hybrid type (Hy-type) glycan product. In combination with alanine scanning mutagenesis, molecular docking calculations and enzymatic activity measurements conducted on a chemically engineered monoclonal antibody substrate unveil two mechanisms for Hy-type recognition and processing by paradigmatic ENGases. Altogether, the experimental data provide pivotal insight into the molecular mechanism of substrate recognition and specificity for GH18 ENGases and further advance our understanding of chemoenzymatic synthesis and remodeling of homogeneous N-glycan glycoproteins.
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Jul 2021
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Stelios
Chrysostomou
,
Rajat
Roy
,
Filippo
Prischi
,
Lucksamon
Thamlikitkul
,
Kathryn L.
Chapman
,
Uwais
Mufti
,
Robert
Peach
,
Laifeng
Ding
,
David
Hancock
,
Christopher
Moore
,
Miriam
Molina-Arcas
,
Francesco
Mauri
,
David J.
Pinato
,
Joel M.
Abrahams
,
Silvia
Ottaviani
,
Leandro
Castellano
,
Georgios
Giamas
,
Jennifer
Pascoe
,
Devmini
Moonamale
,
Sarah
Pirrie
,
Claire
Gaunt
,
Lucinda
Billingham
,
Neil M.
Steven
,
Michael
Cullen
,
David
Hrouda
,
Mathias
Winkler
,
John
Post
,
Philip
Cohen
,
Seth J.
Salpeter
,
Vered
Bar
,
Adi
Zundelevich
,
Shay
Golan
,
Dan
Leibovici
,
Romain
Lara
,
David R.
Klug
,
Sophia N.
Yaliraki
,
Mauricio
Barahona
,
Yulan
Wang
,
Julian
Downward
,
J. Mark
Skehel
,
Maruf M. U.
Ali
,
Michael J.
Seckl
,
Olivier E.
Pardo
Diamond Proposal Number(s):
[9424]
Abstract: Lung and bladder cancers are mostly incurable due to early development of drug resistance and metastatic dissemination. Hence, better therapies that tackle these two processes are urgently needed to improve clinical outcome. We have identified RSK4 as a promoter of drug resistance and metastasis in lung and bladder cancer cells. Silencing this kinase, either through RNA interference or CRISPR, sensitised tumor cells to chemotherapy and hindered metastasis in vitro and in vivo in a tail- vein injection model. Drug screening revealed several floxacin antibiotics as potent RSK4 activation inhibitors and trovafloxacin reproduced all effects of RSK4 silencing in vitro and in/ex vivo using lung cancer xenograft and genetically-engineered mouse models and bladder tumour explants. Through X-ray structure determination and Markov transient and Deuterium exchange analyses, we identified the allosteric binding site and revealed how this compound blocks RSK4 kinase activation through binding to an allosteric site and mimicking a kinase auto-inhibitory mechanism involving the RSK4’s hydrophobic motif. Last, we show that patients undergoing chemotherapy and adhering to prophylactic levofloxacin in the large placebo-controlled randomised phase 3 SIGNIFICANT Trial had significantly (p =0.048) increased long-term overall survival times. Hence, we suggest that RSK4 inhibition may represent an effective therapeutic strategy for treating lung and bladder cancer.
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Jul 2021
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