I03-Macromolecular Crystallography
|
Sarah
Hijazi
,
Francesco
Marchesani
,
Marialaura
Marchetti
,
Valeria
Buoli Comani
,
Paul
Brear
,
Barbara
Campanini
,
Luca
Ronda
,
Serena
Faggiano
,
Eleonora
Gianquinto
,
Somayeh
Asgharpour Hassankiade
,
Barbara
Rolando
,
Francesca
Spyrakis
,
Carlotta
Compari
,
Loretta
Lazzarato
,
Omar
De Bei
,
Emanuela
Frangipani
,
Stefano
Bettati
Diamond Proposal Number(s):
[25402]
Open Access
Abstract: Infections caused by Staphylococcus aureus depend on its ability to access essential nutrients, including acquiring iron from human hemoglobin (Hb) through the iron-regulated surface determinant (Isd) system. The compound 4-[(2-{[5-(1H-indol-3-yl)-1,3,4-oxadiazol-2-yl]sulfanyl}acetyl)amino]benzoic acid (C35) was recently identified as a promising antimicrobial agent for its ability to bind Hb and hamper its interaction with the staphylococcal hemophore IsdB in vitro. Here, we show that C35 inhibits S. aureus growth by targeting the hemophore-driven iron-acquisition system, highlighting its potential as an inhibitor and validating hemophores as antibacterial targets. Furthermore, for drug design purposes, we solved the X-ray structure of Hb:C35 complex. In contrast to the predicted binding pose, C35 binds tetrameric Hb in a cleft between the α subunits, stabilizing a relaxed conformation (R2) and increasing Hb oxygen affinity. This serendipitous result hints to C35 as a promising scaffold for developing compounds with diverse, or even dual, therapeutic aims, with antimicrobial and Hb-modulating activity.
|
May 2026
|
|
I24-Microfocus Macromolecular Crystallography
|
Natalia
Venetz-Arenas
,
Tim
Schulte
,
Sandra
Müller
,
Karin
Wallden
,
Stefanie
Fischer
,
Tom
Resink
,
Nadir
Kadri
,
Maria
Paladino
,
Nicole
Pina
,
Filip
Radom
,
Denis
Villemagne
,
Sandra
Bruckmaier
,
Andreas
Cornelius
,
Tanja
Hospodarsch
,
Evren
Alici
,
Hans-Gustaf
Ljunggren
,
Benedict J.
Chambers
,
Xiao
Han
,
Renhua
Sun
,
Marta
Carroni
,
Victor
Levitsky
,
Tatyana
Sandalova
,
Marcel
Walser
,
Adnane
Achour
Diamond Proposal Number(s):
[21625]
Open Access
Abstract: The balance between affinity and specificity in T cell receptor (TCR)-dependent targeting of HLA-restricted tumor-associated antigens presents a significant challenge for immunotherapy development. T cell engagers that circumvent these limitations are therefore of particular interest. We established a process to generate bispecific Designed Ankyrin Repeat Proteins (DARPins) that simultaneously target HLA-I/peptide complexes and CD3e. These high-affinity T cell engagers elicited CD8+ T cell activation against tumor targets with strong peptide specificity, as confirmed by X-scanning mutagenesis and functional killing assays. A cryo-EM structure of the ternary DARPin/HLA-A*0201/NY-ESO1157-165 complex revealed a rigid, concave DARPin surface spanning the full length of the peptide-binding cleft, contacting both α-helices and the peptide. The present findings reveal promising immuno-oncotherapeutic approaches and demonstrate the feasibility of rapidly developing DARPins with high affinity and specificity for HLA/peptide targets, which can be readily combined with a new generation of anti-CD3e-specific DARPins.
|
Nov 2025
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
|
Krishna M.
Padmanabha Das
,
Jun
Chen
,
Paul S.
Charifson
,
Jeremy
Green
,
Henry
Tang
,
Sanjay
Panchal
,
Fan
Pu
,
Alla
Korepanova
,
Abhinav
Dubey
,
Gustavo
Afanador
,
Vladimir
Stojkovic
,
Boguslaw
Nocek
,
Lance
Bigelow
,
Sarah H.
Stubbs
,
Robert A.
Davey
,
David A.
Degoey
,
Haribabu
Arthanari
,
Mark N.
Namchuk
Diamond Proposal Number(s):
[32394]
Open Access
Abstract: SARS-CoV-2 Mpro is a cysteine protease, that acts as a symmetrical dimer and displays positive cooperativity for substrate turnover. A series of potent reversible covalent peptidomimetic aldehydes and nitriles was designed as Mpro inhibitors. To better understand the observed structure activity relationships (SAR) binding potency and mechanism was examined by enzyme activity assay, surface plasmon resonance, X-ray crystallography, matrix-assisted laser desorption electrospray ionization and NMR. Potent aldehydes bind Mpro cooperativity but bind covalently to only one subunit of the dimer. The analogous nitriles do not bind cooperatively, and the degree of covalent binding observed varied depending on the assay method employed. The NMR studies support that potent inhibition of Mpro by the nitriles does not require covalent binding. The data highlight the caveats in using orthogonal assays to confirm compound mechanism, particularly in cooperative systems.
|
May 2025
|
|
B18-Core EXAFS
|
Diamond Proposal Number(s):
[36367]
Open Access
Abstract: Dry reforming of methane (DRM) offers a sustainable route to convert CH4 and CO2 into syngas, addressing both greenhouse gas emissions and energy demand. However, catalyst deactivation due to sintering and coking limits practical applications. In this work, we developed a mesoporous Ni-based catalyst (Ni/ZrSBA-15-OH) featuring abundant Ni-ZrO2 interfaces and small Ni nanoparticles (5.6 nm) confined within a stable silica framework. This catalyst showed excellent performance, achieving 80% CH4 and 87% CO2 conversions at 750 °C, with minimal coke formation (0.4 mg gcat-1 h-1) and high durability (1.3% CH4 conversion loss over 20 hours). Advanced characterizations (XAS, TEM, H2-TPR, and TPSR) revealed that the metal-oxide interface enhances the activation of reactants and stabilizes active sites. DFT calculations confirmed that the Ni-ZrO2 interface increases the energy barrier for CH* dehydrogenation, effectively suppressing carbon deposition. This study provides a rational strategy for designing structurally robust and coke-resistant Ni-based catalysts for efficient DRM.
|
May 2025
|
|
I03-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[28534]
Open Access
Abstract: The prolyl-tRNA synthetase (PRS) is an essential enzyme for protein translation and a validated target against malaria parasite. We describe five ATP mimetics (L95, L96, L97, L35, and L36) against PRS, exhibiting enhanced thermal stabilities in co-operativity with L-proline. L35 displays the highest thermal stability akin to halofuginone, an established inhibitor of Plasmodium falciparum PRS. Four compounds exhibit nanomolar inhibitory potency against PRS. L35 exhibits the highest potency of ∼1.6 nM against asexual-blood-stage (ABS) and ∼100-fold (EC50) selectivity for the parasite. The macromolecular structures of PfPRS with L95 and L97 in complex with L-pro reveal their binding modes and catalytic site malleability. Arg401 of PfPRS oscillates between two rotameric configurations when in complex with L95, whereas it is locked in one of the configurations due to the larger size of L97. Harnessing such specific and selective chemical features holds significant promise for designing potential inhibitors and expediting drug development efforts.
|
May 2024
|
|
Krios I-Titan Krios I at Diamond
Krios III-Titan Krios III at Diamond
Krios IV-Titan Krios IV at Diamond
|
Tao
Ni
,
Luiza
Mendonca
,
Yanan
Zhu
,
Andrew
Howe
,
Julika
Radecke
,
Pranav M.
Shah
,
Yuewen
Sheng
,
Anna-Sophia
Krebs
,
Helen M. E.
Duyvesteyn
,
Elizabeth
Allen
,
Teresa
Lambe
,
Cameron
Bisset
,
Alexandra
Spencer
,
Susan
Morris
,
David I.
Stuart
,
Sarah
Gilbert
,
Peijun
Zhang
Diamond Proposal Number(s):
[26987]
Open Access
Abstract: Vaccines against SARS-CoV-2 have been proven to be an effective means of decreasing COVID-19 mortality, hospitalization rates, and transmission. One of the vaccines deployed worldwide is ChAdOx1 nCoV-19, which uses an adenovirus vector to drive the expression of the original SARS-CoV-2 spike on the surface of transduced cells. Using cryo-electron tomography and subtomogram averaging, we determined the native structures of the vaccine product expressed on cell surfaces in situ. We show that ChAdOx1-vectored vaccines expressing the Beta SARS-CoV-2 variant produce abundant native prefusion spikes predominantly in one-RBD-up conformation. Furthermore, the ChAdOx1 vectored HexaPro stabilized spike yields higher cell surface expression, enhanced RBD exposure, and reduced shedding of S1 compared to the wild-type. We demonstrate in situ structure determination as a powerful means for studying antigen design options in future vaccine development against emerging novel SARS-CoV-2 variants and broadly against other infectious viruses.
|
Sep 2023
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
|
Kevin P.
Guay
,
Roberta
Ibba
,
John L.
Kiappes
,
Snežana
Vasiljević
,
Francesco
Bonì
,
Maria
De Benedictis
,
Ilaria
Zeni
,
James D.
Le Cornu
,
Mario
Hensen
,
Anu V.
Chandran
,
Anastassia L.
Kantsadi
,
Alessandro T.
Caputo
,
Juan I.
Blanco Capurro
,
Yusupha
Bayo
,
Johan C.
Hill
,
Kieran
Hudson
,
Andrea
Lia
,
Juliane
Brun
,
Stephen G.
Withers
,
Marcelo
Martí
,
Emiliano
Biasini
,
Angelo
Santino
,
Matteo
De Rosa
,
Mario
Milani
,
Carlos P.
Modenutti
,
Daniel N.
Hebert
,
Nicole
Zitzmann
,
Pietro
Roversi
Diamond Proposal Number(s):
[19758]
Open Access
Abstract: Misfolded glycoprotein recognition and endoplasmic reticulum (ER) retention are mediated by the ER glycoprotein folding Quality Control (ERQC) checkpoint enzyme, UDP-Glucose glycoprotein glucosyltransferase (UGGT). UGGT modulation is a promising strategy for broad-spectrum antivirals, rescue-of-secretion therapy in rare disease caused by responsive mutations in glycoprotein genes, and many cancers, but to date no selective UGGT inhibitors are known. The small molecule 5-[(morpholin-4-yl)methyl]quinolin-8-ol (5M-8OH-Q) binds a CtUGGTGT24 ‘WY’ conserved surface motif conserved across UGGTs but not present in other GT24 family glycosyltransferases. 5M-8OH-Q has a 47 μM binding affinity for CtUGGTGT24 in vitro as measured by ligand-enhanced fluorescence. In cellula, 5M-8OH-Q inhibits both human UGGT isoforms at concentrations higher than 750 μM. 5M-8OH-Q binding to CtUGGTGT24 appears to be mutually exclusive to M5-9 glycan binding in an in vitro competition experiment. A medicinal program based on 5M-8OH-Q will yield the next generation of UGGT inhibitors.
|
Sep 2023
|
|
I03-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[9475]
Open Access
Abstract: Gram-negative bacteria contain an asymmetric outer membrane, in which the outer leaflet is composed of lipopolysaccharide (LPS). LPS, a drug target of polymyxin, plays an essential role in drug resistance, biofilm formation and pathogenesis. An important inner membrane protein, YciM, may be responsible for the regulation of LPS biosynthesis and transport. Here we report the crystal structure of YciM from Salmonella typhimurium in a complex with a non-specifically bond molecule, an ethylene glycol, which identified a tunnel that could bind lipids. Our in vitro assays showed that YciM could bind lipid molecules with affinity in the micromolar range, while mutagenic and functional studies confirmed that lipid-binding residues are critical for the function of YciM. Additionally, our data also showed that YciM accurately regulates LPS biosynthesis and transport with YciS, which could help to better understand the regulation mechanism of LPS.
|
Aug 2022
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[8997]
Open Access
Abstract: Bacteriocins are a distinct family of antimicrobial proteins postulated to porate bacterial membranes. However, direct experimental evidence of pore formation by these proteins is lacking. Here we report a multi-mode poration mechanism induced by four-helix bacteriocins, epidermicin NI01 and aureocin A53. Using a combination of crystallography, spectroscopy, bioassays, and nanoscale imaging, we established that individual two-helix segments of epidermicin retain antibacterial activity but each of these segments adopts a particular poration mode. In the intact protein these segments act synergistically to balance out antibacterial and hemolytic activities. The study sets a precedent of multi-mode membrane disruption advancing the current understanding of structure-activity relationships in pore-forming proteins.
|
Aug 2020
|
|
B21-High Throughput SAXS
I03-Macromolecular Crystallography
|
Open Access
Abstract: Mislocalisation, cleavage and aggregation of the human protein TDP-43 is found in many neurodegenerative diseases. As is the case with many other proteins that are completely or partially structurally disordered, production of full-length recombinant TDP-43 in the quantities necessary for structural characterisation has proven difficult. We show that the full-length TDP-43 protein and two truncated N-terminal constructs 1-270 and 1-263 can be heterologously expressed in E. coli. Full-length TDP-43 could be prevented from aggregation during purification using a detergent. Crystals grown from an N-terminal construct (1-270) revealed only the NTD domain (residues 1-80) with molecules arranged as parallel spirals with neighbouring molecules arranged in head-to-tail fashion. In order to obtain detergent free, full-length TDP-43 we mutated all six tryptophan residues that provided low-resolution structure of full-length TDP-43 by small angle X-ray scattering and refining the relative positions of individual domains and intrinsically disordered regions in the model using molecular dynamics.
|
May 2020
|
|
