NONE-No attached Diamond beamline
|
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
|
|
VMXm-Versatile Macromolecular Crystallography microfocus
|
Leila T.
Alexander
,
Janani
Durairaj
,
Andriy
Kryshtafovych
,
Luciano A.
Abriata
,
Yusupha
Bayo
,
Gira
Bhabha
,
Cécile
Breyton
,
Simon G.
Caulton
,
James
Chen
,
Séraphine
Degroux
,
Damian C.
Ekiert
,
Benedikte S.
Erlandsen
,
Peter L.
Freddolino
,
Dominic
Gilzer
,
Chris
Greening
,
Jonathan M.
Grimes
,
Rhys
Grinter
,
Manickam
Gurusaran
,
Marcus D.
Hartmann
,
Charlie J.
Hitchman
,
Jeremy R.
Keown
,
Ashleigh
Kropp
,
Petri
Kursula
,
Andrew L.
Lovering
,
Bruno
Lemaitre
,
Andrea
Lia
,
Shiheng
Liu
,
Maria
Logotheti
,
Shuze
Lu
,
Sigurbjorn
Markusson
,
Mitchell D.
Miller
,
George
Minasov
,
Hartmut H.
Niemann
,
Felipe
Opazo
,
George N.
Phillips
,
Owen R.
Davies
,
Samuel
Rommelaere
,
Monica
Rosas‐lemus
,
Pietro
Roversi
,
Karla
Satchell
,
Nathan
Smith
,
Mark A.
Wilson
,
Kuan‐lin
Wu
,
Xian
Xia
,
Han
Xiao
,
Wenhua
Zhang
,
Z. Hong
Zhou
,
Krzysztof
Fidelis
,
Maya
Topf
,
John
Moult
,
Torsten
Schwede
Diamond Proposal Number(s):
[19946, 23570, 27314, 28534]
Open Access
Abstract: We present an in-depth analysis of selected CASP15 targets, focusing on their biological and functional significance. The authors of the structures identify and discuss key protein features and evaluate how effectively these aspects were captured in the submitted predictions. While the overall ability to predict three-dimensional protein structures continues to impress, reproducing uncommon features not previously observed in experimental structures is still a challenge. Furthermore, instances with conformational flexibility and large multimeric complexes highlight the need for novel scoring strategies to better emphasize biologically relevant structural regions. Looking ahead, closer integration of computational and experimental techniques will play a key role in determining the next challenges to be unraveled in the field of structural molecular biology.
|
Jul 2023
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
|
Diamond Proposal Number(s):
[13385, 14379, 19758]
Open Access
Abstract: None of the current data processing pipelines for X-ray crystallography fragment-based lead discovery (FBLD) consults all the information available when deciding on the lattice and symmetry (i.e., the polymorph) of each soaked crystal. Often, X-ray crystallography FBLD pipelines either choose the polymorph based on cell volume and point-group symmetry of the X-ray diffraction data or leave polymorph attribution to manual intervention on the part of the user. Thus, when the FBLD crystals belong to more than one crystal polymorph, the discovery pipeline can be plagued by space group ambiguity, especially if the polymorphs at hand are variations of the same lattice and, therefore, difficult to tell apart from their morphology and/or their apparent crystal lattices and point groups. In the course of a fragment-based lead discovery effort aimed at finding ligands of the catalytic domain of UDP–glucose glycoprotein glucosyltransferase (UGGT), we encountered a mixture of trigonal crystals and pseudotrigonal triclinic crystals—with the two lattices closely related. In order to resolve that polymorphism ambiguity, we have written and described here a series of Unix shell scripts called CoALLA (crystal polymorph and ligand likelihood-based assignment). The CoALLA scripts are written in Unix shell and use autoPROC for data processing, CCP4-Dimple/REFMAC5 and BUSTER for refinement, and RHOFIT for ligand docking. The choice of the polymorph is effected by carrying out (in each of the known polymorphs) the tasks of diffraction data indexing, integration, scaling, and structural refinement. The most likely polymorph is then chosen as the one with the best structure refinement Rfree statistic. The CoALLA scripts further implement a likelihood-based ligand assignment strategy, starting with macromolecular refinement and automated water addition, followed by removal of the water molecules that appear to be fitting ligand density, and a final round of refinement after random perturbation of the refined macromolecular model, in order to obtain unbiased difference density maps for automated ligand placement. We illustrate the use of CoALLA to discriminate between H3 and P1 crystals used for an FBLD effort to find fragments binding to the catalytic domain of Chaetomium thermophilum UGGT.
|
Dec 2022
|
|
I24-Microfocus Macromolecular Crystallography
|
Open Access
Abstract: 14-3-3 proteins are an important family of hub proteins that play important roles in many cellular processes via a large network of interactions with partner proteins. Many of these protein–protein interactions (PPI) are implicated in human diseases such as cancer and neurodegeneration. The stabilisation of selected 14-3-3 PPIs using drug-like ‘molecular glues’ is a novel therapeutic strategy with high potential. However, the examples reported to date have a number of drawbacks in terms of selectivity and potency. Here, we report that WR-1065, the active species of the approved drug amifostine, covalently modifies 14-3-3σ at an isoform-unique cysteine residue, Cys38. This modification leads to isoform-specific stabilisation of two 14-3-3σ PPIs in a manner that is cooperative with a well characterised molecular glue, fusicoccin A. Our findings reveal a novel stabilisation mechanism for 14-3-3σ, an isoform with particular involvement in cancer pathways. This mechanism can be exploited to harness the enhanced potency conveyed by covalent drug molecules and dual ligand cooperativity. This is demonstrated in two cancer cell lines whereby the cooperative behaviour of fusicoccin A and WR-1065 leads to enhanced efficacy for inducing cell death and attenuating cell growth.
|
Sep 2021
|
|
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Carlos P.
Modenutti
,
Juan I.
Blanco Capurro
,
Roberta
Ibba
,
Dominic S.
Alonzi
,
Mauro N.
Song
,
Snežana
Vasiljević
,
Abhinav
Kumar
,
Anu V.
Chandran
,
Gabor
Tax
,
Lucia
Marti
,
Johan C.
Hill
,
Andrea
Lia
,
Mario
Hensen
,
Thomas
Waksman
,
Jonathan
Rushton
,
Simone
Rubichi
,
Angelo
Santino
,
Marcelo A.
Martí
,
Nicole
Zitzmann
,
Pietro
Roversi
Diamond Proposal Number(s):
[12346, 18069]
Open Access
Abstract: UDP-glucose:glycoprotein glucosyltransferase (UGGT) flags misfolded glycoproteins for ER retention. We report crystal structures of full-length Chaetomium thermophilum UGGT (CtUGGT), two CtUGGT double-cysteine mutants, and its TRXL2 domain truncation (CtUGGT-ΔTRXL2). CtUGGT molecular dynamics (MD) simulations capture extended conformations and reveal clamping, bending, and twisting inter-domain movements. We name “Parodi limit” the maximum distance on the same glycoprotein between a site of misfolding and an N-linked glycan that can be reglucosylated by monomeric UGGT in vitro, in response to recognition of misfold at that site. Based on the MD simulations, we estimate the Parodi limit as around 70–80 Å. Frequency distributions of distances between glycoprotein residues and their closest N-linked glycosylation sites in glycoprotein crystal structures suggests relevance of the Parodi limit to UGGT activity in vivo. Our data support a “one-size-fits-all adjustable spanner” UGGT substrate recognition model, with an essential role for the UGGT TRXL2 domain.
|
Dec 2020
|
|
I03-Macromolecular Crystallography
|
Open Access
Abstract: Background: n-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyses the reversible NAD+-dependent oxidative phosphorylation of n-glyceraldehyde-3-phosphate to 1,3-diphospho-n-glycerate in both glycolysis and gluconeogenesis.
Methods: Four distinct crystal structures of human n-Glyceraldehyde-3-phosphate dehydrogenase (HsGAPDH) have been determined from protein purified from the supernatant of HEK293F human epithelial kidney cells.
Results: X-ray crystallography and mass-spectrometry indicate that the catalytic cysteine of the protein (HsGAPDH Cys152) is partially oxidised to cysteine S-sulfonic acid. The average occupancy for the Cys152-S-sulfonic acid modification over the 20 crystallographically independent copies of HsGAPDH across three of the crystal forms obtained is 0.31±0.17.
Conclusions: The modification induces no significant structural changes on the tetrameric enzyme, and only makes aspecific contacts to surface residues in the active site, in keeping with the hypothesis that the oxidising conditions of the secreted mammalian cell expression system result in HsGAPDH catalytic cysteine S-sulfonic acid modification and irreversible inactivation of the enzyme.
|
Jun 2020
|
|
I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
|
Kelly
Warfield
,
Dominic S.
Alonzi
,
Johan C.
Hill
,
Alessandro T.
Caputo
,
Pietro
Roversi
,
John I.
Kiappes
,
Nicholas
Sheets
,
Matthew
Duchars
,
Raymond
Dwek
,
Julia
Biggins
,
Dale L.
Barnard
,
Sujan
Shresta
,
Anthony
Treston
,
Nicole
Zitzmann
Diamond Proposal Number(s):
[12346]
Abstract: Influenza and dengue viruses present a growing global threat to public health. Both viruses depend on the host endoplasmic reticulum (ER) glycoprotein folding pathway. In 2014, Sadat et al. reported two siblings with a rare genetic defect in ER alpha-glucosidase I (ER Glu I) who showed resistance to viral infections, identifying ER Glu I as a key antiviral target. Here we show that a single dose of UV-4B (the hydrochloride salt form of N-(9′-methoxynonyl)-1- deoxynojirimycin; MON-DNJ) capable of inhibiting Glu I in vivo is sufficient to prevent death in mice infected with lethal viral doses, even when treatment is started as late as 48 hours post-infection. The first crystal structure of mammalian ER Glu I will constitute the basis for the development of potent and selective inhibitors. Targeting ER Glu I with UV-4B derived compounds may alter treatment paradigms for acute viral disease through development of a single-dose therapeutic regime.
|
Mar 2020
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
Krios I-Titan Krios I at Diamond
|
Pietro
Roversi
,
Lucia
Marti
,
Alessandro T.
Caputo
,
Dominic S.
Alonzi
,
Johan C.
Hill
,
Kyle C.
Dent
,
Abhinav
Kumar
,
Mikail D.
Levasseur
,
Andrea
Lia
,
Thomas
Waksman
,
Souradeep
Basu
,
Yentli Soto
Albrecht
,
Kristin
Qian
,
James Patrick
Mcivor
,
Colette B.
Lipp
,
Dritan
Siliqi
,
Snezana
Vasilijevic
,
Shabaz
Mohammed
,
Petra
Lukacik
,
Martin A.
Walsh
,
Angelo
Santino
,
Nicole
Zitzmann
Abstract: Glycoproteins traversing the eukaryotic secretory pathway begin life in the endoplasmic reticulum (ER), where their folding is surveyed by the 170-kDa UDP-glucose:glycoprotein glucosyltransferase (UGGT). The enzyme acts as the single glycoprotein folding quality control checkpoint: it selectively reglucosylates misfolded glycoproteins, promotes their association with ER lectins and associated chaperones, and prevents premature secretion from the ER. UGGT has long resisted structural determination and sequence-based domain boundary prediction. Questions remain on how this single enzyme can flag misfolded glycoproteins of different sizes and shapes for ER retention and how it can span variable distances between the site of misfold and a glucose-accepting N-linked glycan on the same glycoprotein. Here, crystal structures of a full-length eukaryotic UGGT reveal four thioredoxin-like (TRXL) domains arranged in a long arc that terminates in two β-sandwiches tightly clasping the glucosyltransferase domain. The fold of the molecule is topologically complex, with the first β-sandwich and the fourth TRXL domain being encoded by nonconsecutive stretches of sequence. In addition to the crystal structures, a 15-Å cryo-EM reconstruction reveals interdomain flexibility of the TRXL domains. Double cysteine point mutants that engineer extra interdomain disulfide bridges rigidify the UGGT structure and exhibit impaired activity. The intrinsic flexibility of the TRXL domains of UGGT may therefore endow the enzyme with the promiscuity needed to recognize and reglucosylate its many different substrates and/or enable reglucosylation of N-linked glycans situated at variable distances from the site of misfold.
|
Jun 2017
|
|
I02-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Jiangyu
Yan
,
Thomas R.
Beattie
,
Adriana L.
Rojas
,
Kelly
Schermerhorn
,
Tamzin
Gristwood
,
Jonathan C.
Trinidad
,
Sonja V.
Albers
,
Pietro
Roversi
,
Andrew F.
Gardner
,
Nicola G. A.
Abrescia
,
Stephen D.
Bell
Diamond Proposal Number(s):
[9063, 10130]
Open Access
Abstract: Since their initial characterization over 30 years ago, it has been believed that the archaeal B-family DNA polymerases are single-subunit enzymes. This contrasts with the multi-subunit B-family replicative polymerases of eukaryotes. Here we reveal that the highly studied PolB1 from Sulfolobus solfataricus exists as a heterotrimeric complex in cell extracts. Two small subunits, PBP1 and PBP2, associate with distinct surfaces of the larger catalytic subunit and influence the enzymatic properties of the DNA polymerase. Thus, multi-subunit replicative DNA polymerase holoenzymes are present in all three domains of life. We reveal the architecture of the assembly by a combination of cross-linking coupled with mass spectrometry, X-ray crystallography and single-particle electron microscopy. The small subunits stabilize the holoenzyme assembly and the acidic tail of one small subunit mitigates the ability of the enzyme to perform strand-displacement synthesis, with important implications for lagging strand DNA synthesis.
|
May 2017
|
|
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
|
Alessandro T.
Caputo
,
Dominic S.
Alonzi
,
Lucia
Marti
,
Ida-Barbara
Reca
,
John
Kiappes
,
Weston B.
Struwe
,
Alice
Cross
,
Souradeep
Basu
,
Ed
Lowe
,
Benoit
Darlot
,
Angelo
Santino
,
Pietro
Roversi
,
Nicole
Zitzmann
Diamond Proposal Number(s):
[9306]
Abstract: The biosynthesis of enveloped viruses depends heavily on the host cell endoplasmic reticulum (ER) glycoprotein quality control (QC) machinery. This dependency exceeds the dependency of host glycoproteins, offering a window for the targeting of ERQC for the development of broad-spectrum antivirals. We determined small-angle X-ray scattering (SAXS) and crystal structures of the main ERQC enzyme, ER α-glucosidase II (α-GluII; from mouse), alone and in complex with key ligands of its catalytic cycle and antiviral iminosugars, including two that are in clinical trials for the treatment of dengue fever. The SAXS data capture the enzyme’s quaternary structure and suggest a conformational rearrangement is needed for the simultaneous binding of a monoglucosylated glycan to both subunits. The X-ray structures with key catalytic cycle intermediates highlight that an insertion between the +1 and +2 subsites contributes to the enzyme’s activity and substrate specificity, and reveal that the presence of d-mannose at the +1 subsite renders the acid catalyst less efficient during the cleavage of the monoglucosylated substrate. The complexes with iminosugar antivirals suggest that inhibitors targeting a conserved ring of aromatic residues between the α-GluII +1 and +2 subsites would have increased potency and selectivity, thus providing a template for further rational drug design.
|
Aug 2016
|
|
