I24-Microfocus Macromolecular Crystallography
|
Diamond Proposal Number(s):
[20229]
Open Access
Abstract: C1GalT1 is an essential inverting glycosyltransferase responsible for synthesizing the core 1 structure, a common precursor for mucin-type O-glycans found in many glycoproteins. To date, the structure of C1GalT1 and the details of substrate recognition and catalysis remain unknown. Through biophysical and cellular studies, including X-ray crystallography of C1GalT1 complexed to a glycopeptide, we report that C1GalT1 is an obligate GT-A fold dimer that follows a SN2 mechanism. The binding of the glycopeptides to the enzyme is mainly driven by the GalNAc moiety while the peptide sequence provides optimal kinetic and binding parameters. Interestingly, to achieve glycosylation, C1GalT1 recognizes a high-energy conformation of the α-GalNAc-Thr linkage, negligibly populated in solution. By imposing this 3D-arrangement on that fragment, characteristic of α-GalNAc-Ser peptides, C1GalT1 ensures broad glycosylation of both acceptor substrates. These findings illustrate a structural and mechanistic blueprint to explain glycosylation of multiple acceptor substrates, extending the repertoire of mechanisms adopted by glycosyltransferases.
|
May 2022
|
|
I03-Macromolecular Crystallography
|
Itxaso
Anso
,
Luis G. M.
Basso
,
Lei
Wang
,
Alberto
Marina
,
Edgar D.
Páez-Pérez
,
Christian
Jäger
,
Floriane
Gavotto
,
Montse
Tersa
,
Sebastian
Perrone
,
F.-Xabier
Contreras
,
Jacques
Prandi
,
Martine
Gilleron
,
Carole L.
Linster
,
Francisco
Corzana
,
Todd L.
Lowary
,
Beatriz
Trastoy
,
Marcelo E.
Guerin
Diamond Proposal Number(s):
[20113]
Open Access
Abstract: Glycolipids are prominent components of bacterial membranes that play critical roles not only in maintaining the structural integrity of the cell but also in modulating host-pathogen interactions. PatA is an essential acyltransferase involved in the biosynthesis of phosphatidyl-myo-inositol mannosides (PIMs), key structural elements and virulence factors of Mycobacterium tuberculosis. We demonstrate by electron spin resonance spectroscopy and surface plasmon resonance that PatA is an integral membrane acyltransferase tightly anchored to anionic lipid bilayers, using a two-helix structural motif and electrostatic interactions. PatA dictates the acyl chain composition of the glycolipid by using an acyl chain selectivity “ruler.” We established this by a combination of structural biology, enzymatic activity, and binding measurements on chemically synthesized nonhydrolyzable acyl–coenzyme A (CoA) derivatives. We propose an interfacial catalytic mechanism that allows PatA to acylate hydrophobic PIMs anchored in the inner membrane of mycobacteria, through the use of water-soluble acyl-CoA donors.
|
Oct 2021
|
|
I03-Macromolecular Crystallography
|
Francisco
Corzana
,
Ramon
Hurtado-Guerrero
,
Javier
Macías-León
,
Iris A.
Bermejo
,
Alicia
Asín
,
Ana
García-García
,
Ismael
Compañón
,
Ester
Jiménez-Moreno
,
Helena
Coelho
,
Vincenzo
Mangini
,
Inês S.
Albuquerque
,
Filipa
Marcelo
,
Juan Luis
Asensio
,
Gonçalo J. L.
Bernardes
,
Hiren J.
Joshi
,
Roberto
Fiammengo
,
Ola
Blixt
Abstract: The molecular basis of antibody 5E5, which recognizes the entire GalNAc unit as a primary epitope is disclosed. The antibody's contacts with the peptide are limited to mostly two residues, allowing the antibody to show some degree of promiscuity. These findings open the door to the chemical design of peptide-mimetics for developing efficient anti-cancer vaccines and diagnostic tools.
|
Nov 2020
|
|
I04-Macromolecular Crystallography
|
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.
|
Apr 2020
|
|
I24-Microfocus Macromolecular Crystallography
|
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.
|
Feb 2020
|
|
I24-Microfocus Macromolecular Crystallography
|
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.
|
Jan 2020
|
|
I03-Macromolecular Crystallography
|
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.
|
Sep 2018
|
|
I04-Macromolecular Crystallography
|
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.
|
Mar 2018
|
|
I03-Macromolecular Crystallography
|
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.
|
Dec 2017
|
|
I04-Macromolecular Crystallography
|
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]
Open Access
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.
|
Nov 2017
|
|
