I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9948]
Abstract: Understanding the enzyme reaction mechanism can lead to the design of enzyme inhibitors. A Claisen rearrangement was used to allow conversion of an α-1,4-disaccharide into an α-1,3-linked glycosyl carbasugar to target the endo-α-mannosidase from the GH99 glycosidase family, which, unusually, is believed to act through a 1,2-anhydrosugar “epoxide” intermediate. Using NMR and X-ray crystallography, it is shown that glucosyl carbasugar α-aziridines can act as reasonably potent endo-α-mannosidase inhibitors, likely by virtue of their shape mimicry and the interactions of the aziridine nitrogen with the conserved catalytic acid/base of the enzyme active site.
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Nov 2018
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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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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[16609]
Abstract: Overexpression of the Thomsen‐Friedenreich (TF) antigen in cell membrane proteins occurs in 90% of adenocarcinomas. Additionally, the binding of the TF‐antigen to human galectin‐3 (Gal‐3), also frequently overexpressed in malignancy, promotes cancer progression and metastasis. In this context, structures that interfere with this specific interaction display the potential to prevent cancer metastasis. Herein, a multidisciplinary approach, combining the optimized synthesis of a TF‐antigen mimetic with NMR, X‐ray crystallography methods and isothermal titration calorimetry assays has been employed to unravel the molecular structural details that govern the Gal‐3/TF‐mimetic interaction. The TF‐mimetic presents a binding affinity for Gal‐3 similar to the TF‐natural antigen and retains the binding epitope and the bioactive conformation observed for the native antigen. Furthermore, from a thermodynamic perspective a decrease in the enthalpic contribution was observed for the Gal‐3/TF‐mimetic complex, however this behaviour is compensated by a favourable entropy gain. From a structural perspective, these results establish our TF‐mimetic as a scaffold to design multivalent solutions to potentially interfere with Gal‐3 aberrant interactions and likely be used to hamper Gal‐3‐mediated cancer cells adhesion and metastasis.
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Aug 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]
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.
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Nov 2017
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Marija
Petricevic
,
Lukasz F.
Sobala
,
Pearl
Fernandes
,
Lluís
Raich
,
Andrew James
Thompson
,
Ganeko
Bernardo-Seisdedos
,
Oscar
Millet
,
Sha
Zhu
,
Matthieu
Sollogoub
,
Jesús
Jimenez-Barbero
,
Carme
Rovira
,
Gideon J.
Davies
,
Spencer J.
Williams
Diamond Proposal Number(s):
[9948]
Open Access
Abstract: Inhibitor design incorporating features of the reaction coordinate and transition-state structure has emerged as a powerful approach for the development of enzyme inhibitors. Such inhibitors find use as mechanistic probes, chemical biology tools and therapeutics. Endo-α-1,2-mannosidases and endo-α-1,2-mannanases, members of glycoside hydrolase family 99 (GH99), are interesting targets for inhibitor development as they play key roles in N-glycan maturation and microbiotal yeast mannan degradation, respectively. These enzymes are proposed to act via an 1,2-anhydrosugar 'epoxide' mechanism that proceeds through a proposed unusual conformational itinerary. Here, we explore how charge and shape contribute to binding of diverse inhibitors of these enzymes. We report the synthesis of neutral dideoxy, glucal and cyclohexenyl disaccharide inhibitors, their binding to GH99 endo-α-1,2-mannanases, and their structural analysis by X-ray crystallography. Quantum mechanical calculations of the free energy landscapes reveal how the neutral inhibitors provide shape but not charge mimicry of the proposed intermediate and transition state structures. Building upon the knowledge of shape and charge contributions to inhibition of family GH99 enzymes, we design and synthesize α-Man-1,3-noeuromycin, which is revealed to be the most potent (KD 13 nM for Bacteroides xylanisolvens GH99 enzyme) inhibitor of these enzymes yet reported. This work reveals how shape and charge mimicry of transition state features can enable the rational design of potent inhibitors.
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Dec 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
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David
Madariaga
,
Nuria
Martínez-Sáez
,
Víctor J.
Somovilla
,
Helena
Coelho
,
Jessika
Valero-González
,
Jorge
Castro-López
,
Juan L.
Asensio
,
Jesús
Jiménez-Barbero
,
Jesús H.
Busto
,
Alberto
Avenoza
,
Filipa
Marcelo
,
Ramón
Hurtado-Guerrero
,
Francisco
Corzana
,
Jesús M.
Peregrina
Diamond Proposal Number(s):
[8035]
Abstract: Tn antigen (α-O-GalNAc-Ser/Thr) is a convenient cancer biomarker that is recognized by antibodies and lectins. This work yields remarkable results for two plant lectins in terms of epitope recognition and reveals that these receptors show higher affinity for Tn antigen when it is incorporated in the Pro-Asp-Thr-Arg (PDTR) peptide region of mucin MUC1. In contrast, a significant affinity loss is observed when Tn antigen is located in the Ala-His-Gly-Val-Thr-Ser-Ala (AHGVTSA) or Ala-Pro-Gly-Ser-Thr-Ala-Pro (APGSTAP) fragments. Our data indicate that the charged residues, Arg and Asp, present in the PDTR sequence establish noteworthy fundamental interactions with the lectin surface as well as fix the conformation of the peptide backbone, favoring the presentation of the sugar moiety toward the lectin. These results may help to better understand glycopeptide–lectin interactions and may contribute to engineer new binding sites, allowing novel glycosensors for Tn antigen detection to be designed.
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Dec 2014
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