I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Gustavo
Fenalti
,
Nicolas
Villanueva
,
Mark
Griffith
,
Barbra
Pagarigan
,
Sirish Kaushik
Lakkaraju
,
Richard Y.-C.
Huang
,
Nadia
Ladygina
,
Alok
Sharma
,
David
Mikolon
,
Mahan
Abbasian
,
Jeffrey
Johnson
,
Haralambos
Hadjivassiliou
,
Dan
Zhu
,
Philip P.
Chamberlain
,
Ho
Cho
,
Kandasamy
Hariharan
Open Access
Abstract: CD47 is the only 5-transmembrane (5-TM) spanning receptor of the immune system. Its extracellular domain (ECD) is a cell surface marker of self that binds SIRPα and inhibits macrophage phagocytosis, and cancer immuno-therapy approaches in clinical trials are focused on blocking CD47/SIRPα interaction. We present the crystal structure of full length CD47 bound to the function-blocking antibody B6H12. CD47 ECD is tethered to the TM domain via a six-residue peptide linker (114RVVSWF119) that forms an extended loop (SWF loop), with the fundamental role of inserting the side chains of W118 and F119 into the core of CD47 extracellular loop region (ECLR). Using hydrogen-deuterium exchange and molecular dynamics simulations we show that CD47’s ECLR architecture, comprised of two extracellular loops and the SWF loop, creates a molecular environment stabilizing the ECD for presentation on the cell surface. These findings provide insights into CD47 immune recognition, signaling and therapeutic intervention.
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Sep 2021
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[15433]
Open Access
Abstract: The SNM1 nucleases which help maintain genome integrity are members of the metallo-β-lactamase (MBL) structural superfamily. Their conserved MBL-β-CASP-fold SNM1 core provides a molecular scaffold forming an active site which coordinates the metal ions required for catalysis. The features that determine SNM1 endo- versus exonuclease activity, and which control substrate selectivity and binding are poorly understood. We describe a structure of SNM1B/Apollo with two nucleotides bound to its active site, resembling the product state of its exonuclease reaction. The structure enables definition of key SNM1B residues that form contacts with DNA and identifies a 5′ phosphate binding pocket, which we demonstrate is important in catalysis and which has a key role in determining endo- versus exonucleolytic activity across the SNM1 family. We probed the capacity of SNM1B to digest past sites of common endogenous DNA lesions and find that base modifications planar to the nucleobase can be accommodated due to the open architecture of the active site, but lesions axial to the plane of the nucleobase are not well tolerated due to constriction around the altered base. We propose that SNM1B/Apollo might employ its activity to help remove common oxidative lesions from telomeres.
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Aug 2021
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[19301]
Open Access
Abstract: Artemis (SNM1C/DCLRE1C) is an endonuclease that plays a key role in development of B- and T-lymphocytes and in dsDNA break repair by non-homologous end-joining (NHEJ). Artemis is phosphorylated by DNA-PKcs and acts to open DNA hairpin intermediates generated during V(D)J and class-switch recombination. Artemis deficiency leads to congenital radiosensitive severe acquired immune deficiency (RS-SCID). Artemis belongs to a superfamily of nucleases containing metallo-β-lactamase (MBL) and β-CASP (CPSF-Artemis-SNM1-Pso2) domains. We present crystal structures of the catalytic domain of wildtype and variant forms of Artemis, including one causing RS-SCID Omenn syndrome. The catalytic domain of the Artemis has similar endonuclease activity to the phosphorylated full-length protein. Our structures help explain the predominantly endonucleolytic activity of Artemis, which contrasts with the predominantly exonuclease activity of the closely related SNM1A and SNM1B MBL fold nucleases. The structures reveal a second metal binding site in its β-CASP domain unique to Artemis, which is amenable to inhibition by compounds including ebselen. By combining our structural data with that from a recently reported Artemis structure, we were able model the interaction of Artemis with DNA substrates. The structures, including one of Artemis with the cephalosporin ceftriaxone, will help enable the rational development of selective SNM1 nuclease inhibitors.
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Aug 2021
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Krios I-Titan Krios I at Diamond
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Abstract: Persistent infection by oncogenic human papillomavirus (HPV) is the primary cause of cervical cancer, a leading cause of cancer deaths in women worldwide. There are no treatments for HPV infection, and although prophylactic vaccines are effective and safe, they are HPV type specific, provide little therapeutic benefit and developing countries often have limited access to these. Therefore, additional measures against HPV infection are urgently needed. Preventing HPV entry into host cells is an attractive option for therapeutic intervention. The HPV capsid is icosahedral and consists of two proteins, L1 and L2, which participate in entry and infection of host cells. During entry, the virus capsid attaches to the cell surface via binding to heparan sulphate proteoglycans (HSPGs). Cleavage of L2 by a host protease, furin, is necessary for infection and is thought to facilitate a conformational change in the virus capsid. Furin cleavage may affect the ability of HPV to bind to sulphated glycoproteins and a HSPG substitute, heparin. Understanding these proposed structural changes may aid in the development of therapeutics targeting virus entry. Here, we directly visualize the conformation changes to HPV16 pseudovirions (HPV16 PsVs) resulting from cleavage of L2 by exogenous furin using cryoelectron microscopy (cryo-EM). At 5 Å resolution, we observed that furin-cleaved HPV16 PsVs capsids display widespread changes in the arrangement of capsomeres relative to uncleaved control virions. This structural change is relevant because heparin has previously been observed to bind to the HPV16 capsid in the canyon surrounding the capsomere at the five-fold icosahedral symmetry axis, but not in other canyons between capsomeres, related by pseudo-symmetry. This suggests that differences in the relative orientations of the surrounding capsomeres to each other either prevent or allow heparin binding. We observed a narrowing of the putative heparin binding site by 0.4 Å after furin cleavage and propose that this change may be responsible for the transfer of HPV from cell-surface HSPGs to the unknown entry receptor(s) by a yet unidentified mechanism.
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Aug 2021
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I12-JEEP: Joint Engineering, Environmental and Processing
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Diamond Proposal Number(s):
[26476]
Abstract: In this work, three new pharmaceutical salts of fenbendazole (FNB), a benzimidazole-based anthelmintic drug, with sulfonic acids have been obtained and thoroughly investigated by different analytical techniques, including thermal methods, infrared/Raman spectroscopy, and theoretical methods (periodic DFT computations and Bader analyses of the crystalline electronic density). Single-crystal and high-resolution synchrotron powder X-ray diffraction data for the first time made it possible to determine the crystal structures of mesylate and tosylate salts of the drug, which were further validated by dispersion-corrected density functional theory calculations. All the solid forms were stabilized by a robust R22(8) supramolecular motif formed by relatively strong N–H···O hydrogen bonds. In the monohydrate of FNB tosylate, a considerable gain in the stabilization energy was due to the intermolecular interactions generated by the water molecules. A careful examination of the solubility–pH profile of the FNB salts revealed that, despite being thermodynamically unstable within the physiologically relevant pH range, the new solid forms demonstrated superior dissolution performance in terms of both the apparent solubility and the release rate in comparison to the parent drug. Since FNB has also been reported to possess anticancer activity, improving the drug’s poor physicochemical properties through salt formation with the selected sulfonic acids is expected to promote further investigations toward repurposing of this potent compound.
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Jul 2021
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19800]
Abstract: The Notch signaling system links cellular fate to that of its neighbors, driving proliferation, apoptosis, and cell differentiation in metazoans, whereas dysfunction leads to debilitating developmental disorders and cancers. Other than a five-by-five domain complex, it is unclear how the 40 extracellular domains of the Notch1 receptor collectively engage the 19 domains of its canonical ligand, Jagged1, to activate Notch1 signaling. Here, using cross-linking mass spectrometry (XL-MS), biophysical, and structural techniques on the full extracellular complex and targeted sites, we identify five distinct regions, two on Notch1 and three on Jagged1, that form an interaction network. The Notch1 membrane–proximal regulatory region individually binds to the established Notch1 epidermal growth factor (EGF) 8–EGF13 and Jagged1 C2–EGF3 activation sites as well as to two additional Jagged1 regions, EGF8–EGF11 and cysteine-rich domain. XL-MS and quantitative interaction experiments show that the three Notch1-binding sites on Jagged1 also engage intramolecularly. These interactions, together with Notch1 and Jagged1 ectodomain dimensions and flexibility, determined by small-angle X-ray scattering, support the formation of nonlinear architectures. Combined, the data suggest that critical Notch1 and Jagged1 regions are not distal but engage directly to control Notch1 signaling, thereby redefining the Notch1–Jagged1 activation mechanism and indicating routes for therapeutic applications.
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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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I13-2-Diamond Manchester Imaging
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Diamond Proposal Number(s):
[22588, 17241]
Open Access
Abstract: Colonic crypts are tubular glands that multiply through a symmetric branching process called crypt fission. During the early stages of colorectal cancer, the normal fission process is disturbed, leading to asymmetrical branching or budding. The challenging shapes of the budding crypts make it difficult to prepare paraffin sections for conventional histology, resulting in colonic cross sections with crypts that are only partially visible. To study crypt budding in situ and in three dimensions (3D), we employ X-ray micro-computed tomography to image intact colons, and a new method we developed (3D cyclorama) to digitally unroll them. Here, we present, verify and validate our ‘3D cyclorama’ method that digitally unrolls deformed tubes of non-uniform thickness. It employs principles from electrostatics to reform the tube into a series of onion-like surfaces, which are mapped onto planar panoramic views. This enables the study of features extending over several layers of the tube’s depth, demonstrated here by two case studies: (i) microvilli in the human placenta and (ii) 3D-printed adhesive films for drug delivery. Our 3D cyclorama method can provide novel insights into a wide spectrum of applications where digital unrolling or flattening is necessary, including long bones, teeth roots and ancient scrolls.
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Jul 2021
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I03-Macromolecular Crystallography
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Igor M.
Ferreira
,
José Edwin N.
Quesñay
,
Alliny C. S.
Bastos
,
Camila T.
Rodrigues
,
Melanie
Vollmar
,
Tobias
Krojer
,
Claire
Strain-Damerell
,
Nicola A.
Burgess-Brown
,
Frank
Von Delft
,
Wyatt W.
Yue
,
Sandra M G.
Dias
,
Andre L. B.
Ambrosio
Diamond Proposal Number(s):
[8421]
Open Access
Abstract: Cancer cells exhibit an altered metabolic phenotype, consuming higher levels of the amino acid glutamine. This metabolic reprogramming depends on increased mitochondrial glutaminase activity to convert glutamine to glutamate, an essential precursor for bioenergetic and biosynthetic processes in cells. Mammals encode the kidney-type (GLS) and liver-type (GLS2) glutaminase isozymes. GLS is overexpressed in cancer and associated with enhanced malignancy. On the other hand, GLS2 is either a tumor suppressor or an oncogene, depending on the tumor type. The GLS structure and activation mechanism are well known, while the structural determinants for GLS2 activation remain elusive. Here, we describe the structure of the human glutaminase domain of GLS2, followed by the functional characterization of the residues critical for its activity. Increasing concentrations of GLS2 lead to tetramer stabilization, a process enhanced by phosphate. In GLS2, the so-called “lid loop” is in a rigid open conformation, which may be related to its higher affinity for phosphate and lower affinity for glutamine; hence, it has lower glutaminase activity than GLS. The lower affinity of GLS2 for glutamine is also related to its less electropositive catalytic site than GLS, as indicated by a Thr225Lys substitution within the catalytic site decreasing the GLS2 glutamine concentration corresponding to half-maximal velocity (K0.5). Finally, we show that the Lys253Ala substitution (corresponding to the Lys320Ala in the GLS “activation” loop, formerly known as the “gating” loop) renders a highly active protein in stable tetrameric form. We conclude that the “activation” loop, a known target for GLS inhibition, may also be a drug target for GLS2.
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Jun 2021
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I14-Hard X-ray Nanoprobe
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Diamond Proposal Number(s):
[19838, 20548]
Open Access
Abstract: Transition metal complexes are often prodrugs which undergo activation by ligand exchange and redox reactions before they interact with target sites. It is therefore important to understand the roles of both the metal and the ligands in their activation, especially in cells. Here we use a combination of synchrotron nanoprobe X-ray fluorescence (XRF) from Os L3M5 and Br KL3 emissions and inductively coupled plasma-mass spectrometry (ICP-MS) detection of 189Os, 79Br, and 127I, to investigate the time-dependent accumulation and localization of osmium as well as the monodentate ligand and the chelated phenylazopyridine in A2780 human ovarian cancer cells treated with the potent anticancer complexes [Os(η6-p-cymene)(4-R2-phenyl-azopyridine-5-R1)X]PF6, with R2 = NMe2 or OH, R1 = H or Br, and X = Cl or I. The data confirm that the relatively inert iodido complexes are activated rapidly in cancer cells by release of the iodido ligand, probably initiated by attack by the intracellular tripeptide glutathione (γ-L-Glu-l-Cys-Gly) on the azo double bond. The bond between osmium and the azopyridine appears to remain stable in cells for ca. 24 h, although some release of the chelated ligand is observed. Interestingly, the complexes seem to be degraded more rapidly in normal human cells, perhaps providing a possible mechanism for selective cytotoxicity towards cancer cells.
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Jun 2021
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