I04-Macromolecular Crystallography
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Iris A.
Bermejo
,
Claudio D.
Navo
,
Jorge
Castro-lópez
,
Ana
Guerreiro
,
Ester
Jiménez-moreno
,
Elena M.
Sánchez Fernández
,
Fayna
García-martín
,
Hiroshi
Hinou
,
Shin-ichiro
Nishimura
,
José M.
García Fernández
,
Carmen Ortiz
Mellet
,
Alberto
Avenoza
,
Jesús H.
Busto
,
Gonçalo J. L.
Bernardes
,
Ramon
Hurtado-guerrero
,
Jesús M.
Peregrina
,
Francisco
Corzana
Diamond Proposal Number(s):
[10121]
Open Access
Abstract: The Tn antigen (GalNAc-α-1-O-Thr/Ser) is a well-known tumor-associated carbohydrate determinant. The use of glycopeptides that incorporate this structure has become a significant and promising niche of research owing to their potential use as anticancer vaccines. Herein, the conformational preferences of a glycopeptide with an unnatural Tn antigen, characterized by a threonine decorated with an sp2-iminosugar-type α-GalNAc mimic, have been studied both in solution, by combining NMR spectroscopy and molecular dynamics simulations, and in the solid state bound to an anti-mucin-1 (MUC1) antibody, by X-ray crystallography. The Tn surrogate can mimic the main conformer sampled by the natural antigen in solution and exhibits high affinity towards anti-MUC1 antibodies. Encouraged by these data, a cancer vaccine candidate based on this unnatural glycopeptide and conjugated to the carrier protein Keyhole Limpet Hemocyanin (KLH) has been prepared and tested in mice. Significantly, the experiments in vivo have proved that this vaccine elicits higher levels of specific anti-MUC1 IgG antibodies than the analog that bears the natural Tn antigen and that the elicited antibodies recognize human breast cancer cells with high selectivity. Altogether, we compile evidence to confirm that the presentation of the antigen, both in solution and in the bound state, plays a critical role in the efficacy of the designed cancer vaccines. Moreover, the outcomes derived from this vaccine prove that there is room for exploring further adjustments at the carbohydrate level that could contribute to designing more efficient cancer vaccines.
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Apr 2020
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[20229]
Open Access
Abstract: Core-fucosylation is an essential biological modification by which a fucose is transferred from GDP-β-L-fucose to the innermost N-acetylglucosamine residue of N-linked glycans. A single human enzyme α1,6-fucosyltransferase (FUT8) is the only enzyme responsible for this modification via the addition of an α-1,6-linked fucose to N-glycans. To date, the details of substrate recognition and catalysis by FUT8 remain unknown. Here, we report the crystal structure of FUT8 complexed with GDP and a biantennary complex N-glycan (G0), which provides insight into both substrate recognition and catalysis. FUT8 follows an SN2 mechanism and deploys a series of loops and an α-helix which all contribute in forming the binding site. An exosite, formed by one of these loops and an SH3 domain, is responsible for the recognition of branched sugars, making contacts specifically to the α1,3 arm GlcNAc, a feature required for catalysis. This information serves as a framework for inhibitor design, and helps to assess its potential as a therapeutic target.
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Feb 2020
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I24-Microfocus Macromolecular Crystallography
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Matilde
De Las Rivas
,
Earnest James
Paul Daniel
,
Yoshiki
Narimatsu
,
Ismael
Compañón
,
Kentaro
Kato
,
Pablo
Hermosilla
,
Aurélien
Thureau
,
Laura
Ceballos-laita
,
Helena
Coelho
,
Pau
Bernadó
,
Filipa
Marcelo
,
Lars
Hansen
,
Ryota
Maeda
,
Anabel
Lostao
,
Francisco
Corzana
,
Henrik
Clausen
,
Thomas A.
Gerken
,
Ramon
Hurtado-guerrero
Diamond Proposal Number(s):
[14739]
Abstract: Polypeptide GalNAc-transferase T3 (GalNAc-T3) regulates fibroblast growth factor 23 (FGF23) by O-glycosylating Thr178 in a furin proprotein processing motif RHT178R↓S. FGF23 regulates phosphate homeostasis and deficiency in GALNT3 or FGF23 results in hyperphosphatemia and familial tumoral calcinosis. We explored the molecular mechanism for GalNAc-T3 glycosylation of FGF23 using engineered cell models and biophysical studies including kinetics, molecular dynamics and X-ray crystallography of GalNAc-T3 complexed to glycopeptide substrates. GalNAc-T3 uses a lectin domain mediated mechanism to glycosylate Thr178 requiring previous glycosylation at Thr171. Notably, Thr178 is a poor substrate site with limiting glycosylation due to substrate clashes leading to destabilization of the catalytic domain flexible loop. We suggest GalNAc-T3 specificity for FGF23 and its ability to control circulating levels of intact FGF23 is achieved by FGF23 being a poor substrate. GalNAc-T3’s structure further reveals the molecular bases for reported disease-causing mutations. Our findings provide an insight into how GalNAc-T isoenzymes achieve isoenzyme-specific nonredundant functions.
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Jan 2020
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I03-Macromolecular Crystallography
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Matilde
De Las Rivas
,
Earnest James
Paul Daniel
,
Helena
Coelho
,
Erandi
Lira-navarrete
,
Lluis
Raich
,
Ismael
Compañón
,
Ana
Diniz
,
Laura
Lagartera
,
Jesús
Jiménez-barbero
,
Henrik
Clausen
,
Carme
Rovira
,
Filipa
Marcelo
,
Francisco
Corzana
,
Thomas A.
Gerken
,
Ramon
Hurtado-guerrero
Diamond Proposal Number(s):
[10121]
Open Access
Abstract: Mucin-type O-glycosylation is initiated by a family of polypeptide GalNAc-transferases (GalNAc-Ts) which are type-II transmembrane proteins that contain Golgi luminal catalytic and lectin domains that are connected by a flexible linker. Several GalNAc-Ts, including GalNAc-T4, show both long-range and short-range prior glycosylation specificity, governed by their lectin and catalytic domains, respectively. While the mechanism of the lectin-domain-dependent glycosylation is well-known, the molecular basis for the catalytic-domain-dependent glycosylation of glycopeptides is unclear. Herein, we report the crystal structure of GalNAc-T4 bound to the diglycopeptide GAT*GAGAGAGT*TPGPG (containing two α-GalNAc glycosylated Thr (T*), the PXP motif and a “naked” Thr acceptor site) that describes its catalytic domain glycopeptide GalNAc binding site. Kinetic studies of wild-type and GalNAc binding site mutant enzymes show the lectin domain GalNAc binding activity dominates over the catalytic domain GalNAc binding activity and that these activities can be independently eliminated. Surprisingly, a flexible loop protruding from the lectin domain was found essential for the optimal activity of the catalytic domain. This work provides the first structural basis for the short-range glycosylation preferences of a GalNAc-T.
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Sep 2018
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I04-Macromolecular Crystallography
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Ramon
Hurtado-guerrero
,
Matilde
De Las Rivas
,
Helena
Coelho
,
Ana
Diniz
,
Erandi
Lira-navarrete
,
Ismael
Compañón
,
Jesús
Jiménez-barbero
,
Katrine
T. Schjoldager
,
Eric
P. Bennett
,
Sergey
Y. Vakhrushev
,
Henrik
Clausen
,
Francisco
Corzana
,
Filipa
Marcelo
Diamond Proposal Number(s):
[10121]
Abstract: The family of polypeptide GalNAc‐transferases (GalNAc‐Ts) orchestrates the initiating step of mucin‐type protein O‐glycosylation by transfer of GalNAc moieties to serine and threonine residues in proteins. Deficiencies and dysregulation of GalNAc‐T isoenzymes have been found to be related to different diseases. Recently, we have demonstrated that an inactive GalNAc‐T2 mutant (F104S), which is not located at the active site, induces low levels of high‐density lipoprotein cholesterol (HDL‐C) in humans. Here, we have deciphered the molecular basis for F104S mutant inactivation. Saturation transfer difference NMR experiments demonstrate that the mutation induces loss of binding to peptide substrates. The analysis of the crystal structure of the F104S mutant bound to UDP‐GalNAc, combined with molecular dynamics (MD) simulations, has revealed that the flexible loop is disordered and displays larger conformational changes in the mutant enzyme than in the wild‐type (WT) enzyme. 19F‐NMR experiments reveal that the WT enzyme reaches the active state only in the presence of UDP‐GalNAc, providing compelling evidences that GalNAc‐T2 adopts an UDP‐GalNAc‐dependent induced‐fit mechanism. The F104S mutation precludes the enzyme to achieve the active conformation and concomitantly to bind peptide substrates. The present study provides new insights into the catalytic mechanism of the large family of GalNAc‐Ts and how these enzymes orchestrate protein O‐glycosylation.
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Mar 2018
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I03-Macromolecular Crystallography
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Matilde
De Las Rivas
,
Erandi
Lira-navarrete
,
Earnest James Paul
Daniel
,
Ismael
Compañón
,
Helena
Coelho
,
Ana
Diniz
,
Jesús
Jiménez-barbero
,
Jesús M.
Peregrina
,
Henrik
Clausen
,
Francisco
Corzana
,
Filipa
Marcelo
,
Gonzalo
Jiménez-osés
,
Thomas A.
Gerken
,
Ramon
Hurtado-guerrero
Diamond Proposal Number(s):
[10121]
Open Access
Abstract: The polypeptide GalNAc-transferases (GalNAc-Ts), that initiate mucin-type O-glycosylation, consist of a catalytic and a lectin domain connected by a flexible linker. In addition to recognizing polypeptide sequence, the GalNAc-Ts exhibit unique long-range N- and/or C-terminal prior glycosylation (GalNAc-O-Ser/Thr) preferences modulated by the lectin domain. Here we report studies on GalNAc-T4 that reveal the origins of its unique N-terminal long-range glycopeptide specificity, which is the opposite of GalNAc-T2. The GalNAc-T4 structure bound to a monoglycopeptide shows that the GalNAc-binding site of its lectin domain is rotated relative to the homologous GalNAc-T2 structure, explaining their different long-range preferences. Kinetics and molecular dynamics simulations on several GalNAc-T2 flexible linker constructs show altered remote prior glycosylation preferences, confirming that the flexible linker dictates the rotation of the lectin domain, thus modulating the GalNAc-Ts' long-range preferences. This work for the first time provides the structural basis for the different remote prior glycosylation preferences of the GalNAc-Ts.
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Dec 2017
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I04-Macromolecular Crystallography
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Víctor Jesús
Somovilla
,
Iris Alicia
Bermejo
,
Inês S.
Albuquerque
,
Nuria
Martínez-sáez
,
Jorge
Castro-lópez
,
Fayna
Garcia Martin
,
Ismael
Compañón
,
Hiroshi
Hinou
,
Shin-ichiro
Nishimura
,
Jesús
Jiménez-barbero
,
Juan Luis
Asensio
,
Alberto
Avenoza
,
Jesús H
Busto
,
Ramon
Hurtado-guerrero
,
Jesús M.
Peregrina
,
Gonçalo J.l.
Bernardes
,
Francisco
Corzana
Diamond Proposal Number(s):
[10121]
Abstract: A structure-based design of a new generation tumor-associated glycopeptides with improved affinity against two anti-MUC1 antibodies is described. These unique antigens feature a fluorinated proline residue, such as a (4S)-4-fluoro-L-proline or 4,4-difluoroproline, at the most immunogenic domain. Binding assays using bio-layer interferometry reveal 3-fold to 10-fold affinity improvement with respect to the natural (glyco)peptides. According to X-ray crystallography and MD simulations, the fluorinated residues stabilize the antigen-antibody complex by enhancing key CH/π interactions. Interestingly, a notable improvement in detection of cancer-associated anti-MUC1 antibodies from serum of patients with prostate cancer is achieved with the non-natural antigens, which proves that these derivatives can be considered better diagnostic tools than the natural antigen for this type of cancer.
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Nov 2017
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I02-Macromolecular Crystallography
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Jessika
Valero-gonzález
,
Christina
Leonhard-melief
,
Erandi
Lira
,
Gonzalo
Jiménez-osés
,
Cristina
Hernández-ruiz
,
María Carmen
Pallarés
,
Inmaculada
Yruela
,
Deepika
Vasudevan
,
Anabel
Lostao
,
Francisco
Corzana
,
Hideyuki
Takeuchi
,
Robert S
Haltiwanger
,
Ramon
Hurtado-guerrero
Abstract: Protein O-fucosyltransferase 2 (POFUT2) is an essential enzyme that fucosylates serine and threonine residues of folded thrombospondin type 1 repeats (TSRs). To date, the mechanism by which this enzyme recognizes very dissimilar TSRs has been unclear. By engineering a fusion protein, we report the crystal structure of Caenorhabditis elegans POFUT2 (CePOFUT2) in complex with GDP and human TSR1 that suggests an inverting mechanism for fucose transfer assisted by a catalytic base and shows that nearly half of the TSR1 is embraced by CePOFUT2. A small number of direct interactions and a large network of water molecules maintain the complex. Site-directed mutagenesis demonstrates that POFUT2 fucosylates threonine preferentially over serine and relies on folded TSRs containing the minimal consensus sequence C-X-X-S/T-C. Crystallographic and mutagenesis data, together with atomic-level simulations, uncover a binding mechanism by which POFUT2 promiscuously recognizes the structural fingerprint of poorly homologous TSRs through a dynamic network of water-mediated interactions.
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Mar 2016
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Nuria
Martínez-sáez
,
Jorge
Castro-lópez
,
Jessika
Valero-gonzález
,
David
Madariaga
,
Ismael
Compañón
,
Víctor J.
Somovilla
,
Míriam
Salvadó
,
Juan L.
Asensio
,
Jesús
Jiménez-barbero
,
Alberto
Avenoza
,
Jesús H.
Busto
,
Gonçalo J. L.
Bernardes
,
Jesús M.
Peregrina
,
Ramón
Hurtado-guerrero
,
Francisco
Corzana
Diamond Proposal Number(s):
[10121, 8035]
Open Access
Abstract: The structural features of MUC1-like glycopeptides bearing the Tn antigen (α-O-GalNAc-Ser/Thr) in complex with an anti MUC-1 antibody are reported at atomic resolution. For the α-O-GalNAc-Ser derivative, the glycosidic linkage adopts a high-energy conformation, barely populated in the free state. This unusual structure (also observed in an α-S-GalNAc-Cys mimic) is stabilized by hydrogen bonds between the peptidic fragment and the sugar. The selection of a particular peptide structure by the antibody is thus propagated to the carbohydrate through carbohydrate/peptide contacts, which force a change in the orientation of the sugar moiety. This seems to be unfeasible in the α-O-GalNAc-Thr glycopeptide owing to the more limited flexibility of the side chain imposed by the methyl group. Our data demonstrate the non-equivalence of Ser and Thr O-glycosylation points in molecular recognition processes. These features provide insight into the occurrence in nature of the APDTRP epitope for anti-MUC1 antibodies.
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Aug 2015
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Erandi
Lira-navarrete
,
Matilde
De Las Rivas
,
Ismael
Compañón
,
María Carmen
Pallarés
,
Yun
Kong
,
Javier
Iglesias-fernández
,
Gonçalo J. L.
Bernardes
,
Jesús M.
Peregrina
,
Carme
Rovira
,
Pau
Bernadó
,
Pierpaolo
Bruscolini
,
Henrik
Clausen
,
Anabel
Lostao
,
Francisco
Corzana
,
Ramon
Hurtado-guerrero
Diamond Proposal Number(s):
[8035, 10121]
Open Access
Abstract: Protein O-glycosylation is controlled by polypeptide GalNAc-transferases (GalNAc-Ts) that uniquely feature both a catalytic and lectin domain. The underlying molecular basis of how the lectin domains of GalNAc-Ts contribute to glycopeptide specificity and catalysis remains unclear. Here we present the first crystal structures of complexes of GalNAc-T2 with glycopeptides that together with enhanced sampling molecular dynamics simulations demonstrate a cooperative mechanism by which the lectin domain enables free acceptor sites binding of glycopeptides into the catalytic domain. Atomic force microscopy and small-angle X-ray scattering experiments further reveal a dynamic conformational landscape of GalNAc-T2 and a prominent role of compact structures that are both required for efficient catalysis. Our model indicates that the activity profile of GalNAc-T2 is dictated by conformational heterogeneity and relies on a flexible linker located between the catalytic and the lectin domains. Our results also shed light on how GalNAc-Ts generate dense decoration of proteins with O-glycans.
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May 2015
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