I24-Microfocus Macromolecular Crystallography
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Itxaso
Anso
,
Andreas
Naegeli
,
Javier O.
Cifuente
,
Ane
Orrantia
,
Erica
Andersson
,
Olatz
Zenarruzabeitia
,
Alicia
Moraleda-Montoya
,
Mikel
Garcia-Alija
,
Francisco
Corzana
,
Rafael A.
Del Orbe
,
Francisco
Borrego
,
Beatriz
Trastoy
,
Jonathan
Sjögren
,
Marcelo E.
Guerin
Diamond Proposal Number(s):
[28360]
Open Access
Abstract: Red blood cell antigens play critical roles in blood transfusion since donor incompatibilities can be lethal. Recipients with the rare total deficiency in H antigen, the Oh Bombay phenotype, can only be transfused with group Oh blood to avoid serious transfusion reactions. We discover FucOB from the mucin-degrading bacteria Akkermansia muciniphila as an α-1,2-fucosidase able to hydrolyze Type I, Type II, Type III and Type V H antigens to obtain the afucosylated Bombay phenotype in vitro. X-ray crystal structures of FucOB show a three-domain architecture, including a GH95 glycoside hydrolase. The structural data together with site-directed mutagenesis, enzymatic activity and computational methods provide molecular insights into substrate specificity and catalysis. Furthermore, using agglutination tests and flow cytometry-based techniques, we demonstrate the ability of FucOB to convert universal O type into rare Bombay type blood, providing exciting possibilities to facilitate transfusion in recipients/patients with Bombay phenotype.
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Mar 2023
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B21-High Throughput SAXS
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Beatriz
Trastoy
,
Jonathan J.
Du
,
Javier O.
Cifuente
,
Lorena
Rudolph
,
Mikel
Garcia-Alija
,
Erik H.
Klontz
,
Daniel
Deredge
,
Nazneen
Sultana
,
Chau G.
Huynh
,
Maria W.
Flowers
,
Chao
Li
,
Diego E.
Sastre
,
Lai-Xi
Wang
,
Francisco
Corzana
,
Alvaro
Mallagaray
,
Eric J.
Sundberg
,
Marcelo E.
Guerin
Diamond Proposal Number(s):
[1534, 28360]
Open Access
Abstract: Bacterial pathogens have evolved intricate mechanisms to evade the human immune system, including the production of immunomodulatory enzymes. Streptococcus pyogenes serotypes secrete two multi-modular endo-β-N-acetylglucosaminidases, EndoS and EndoS2, that specifically deglycosylate the conserved N-glycan at Asn297 on IgG Fc, disabling antibody-mediated effector functions. Amongst thousands of known carbohydrate-active enzymes, EndoS and EndoS2 represent just a handful of enzymes that are specific to the protein portion of the glycoprotein substrate, not just the glycan component. Here, we present the cryoEM structure of EndoS in complex with the IgG1 Fc fragment. In combination with small-angle X-ray scattering, alanine scanning mutagenesis, hydrolytic activity measurements, enzyme kinetics, nuclear magnetic resonance and molecular dynamics analyses, we establish the mechanisms of recognition and specific deglycosylation of IgG antibodies by EndoS and EndoS2. Our results provide a rational basis from which to engineer novel enzymes with antibody and glycan selectivity for clinical and biotechnological applications.
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Mar 2023
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B21-High Throughput SAXS
I24-Microfocus Macromolecular Crystallography
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Mikel
García-Alija
,
Jonathan J.
Du
,
Izaskun
Ordóñez
,
Asier
Diz-Vallenilla
,
Alicia
Moraleda-Montoya
,
Nazneen
Sultana
,
Chau G.
Huynh
,
Chao
Li
,
Thomas Connor
Donahue
,
Lai-Xi
Wang
,
Beatriz
Trastoy
,
Eric J.
Sundberg
,
Marcelo E.
Guerin
Diamond Proposal Number(s):
[20113]
Open Access
Abstract: Bacteria produce a remarkably diverse range of glycoside hydrolases to metabolize glycans from the environment as a primary source of nutrients, and to promote the colonization and infection of a host. Here we focus on EndoE, a multi-modular glycoside hydrolase secreted by Enterococcus faecalis, one of the leading causes of healthcare-associated infections. We provide X-ray crystal structures of EndoE, which show an architecture composed of four domains, including GH18 and GH20 glycoside hydrolases connected by two consecutive three α-helical bundles. We determine that the GH20 domain is an exo-β-1,2-N-acetylglucosaminidase, whereas the GH18 domain is an endo-β-1,4-N-acetylglucosaminidase that exclusively processes the central core of complex-type or high-mannose-type N-glycans. Both glycoside hydrolase domains act in a concerted manner to process diverse N-glycans on glycoproteins, including therapeutic IgG antibodies. EndoE combines two enzyme domains with distinct functions and glycan specificities to play a dual role in glycan metabolism and immune evasion.
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Mar 2022
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I03-Macromolecular Crystallography
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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.
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Oct 2021
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[20113]
Open Access
Abstract: N-glycosylation is one of the most abundant post-translational modifications of proteins, essential for many physiological processes, including protein folding, protein stability, oligomerization and aggregation, and molecular recognition events. Defects in the N-glycosylation pathway cause diseases that are classified as congenital disorders of glycosylation. The ability to manipulate protein N-glycosylation is critical not only to our fundamental understanding of biology, but also for the development of new drugs for a wide range of human diseases. Chemoenzymatic synthesis using engineered endo-β-N-acetylglucosaminidases (ENGases) has been used extensively to modulate the chemistry of N-glycosylated proteins. However, defining the molecular mechanisms by which ENGases specifically recognize and process N-glycans remains a major challenge. Here we present the X-ray crystal structure of the ENGase EndoBT-3987 from Bacteroides thetaiotaomicron in complex with a hybrid type (Hy-type) glycan product. In combination with alanine scanning mutagenesis, molecular docking calculations and enzymatic activity measurements conducted on a chemically engineered monoclonal antibody substrate unveil two mechanisms for Hy-type recognition and processing by paradigmatic ENGases. Altogether, the experimental data provide pivotal insight into the molecular mechanism of substrate recognition and specificity for GH18 ENGases and further advance our understanding of chemoenzymatic synthesis and remodeling of homogeneous N-glycan glycoproteins.
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Jul 2021
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[20113]
Open Access
Abstract: Akkermansia muciniphila is a mucin-degrading bacterium commonly found in the human gut that promotes a beneficial effect on health, likely based on the regulation of mucus thickness and gut barrier integrity, but also on the modulation of the immune system. In this work, we focus in OgpA from A. muciniphila, an O-glycopeptidase that exclusively hydrolyzes the peptide bond N-terminal to serine or threonine residues substituted with an O-glycan. We determine the high-resolution X-ray crystal structures of the unliganded form of OgpA, the complex with the glycodrosocin O-glycopeptide substrate and its product, providing a comprehensive set of snapshots of the enzyme along the catalytic cycle. In combination with O-glycopeptide chemistry, enzyme kinetics, and computational methods we unveil the molecular mechanism of O-glycan recognition and specificity for OgpA. The data also contribute to understanding how A. muciniphila processes mucins in the gut, as well as analysis of post-translational O-glycosylation events in proteins.
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Sep 2020
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B21-High Throughput SAXS
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Ane
Rodrigo-Unzueta
,
Mattia
Ghirardello
,
Saioa
Urresti
,
Ignacio
Delso
,
David
Giganti
,
Itxaso
Anso
,
Beatriz
Trastoy
,
Natalia
Comino
,
Montse
Tersa
,
Cecilia
D'Angelo
,
Javier O.
Cifuente
,
Alberto
Marina
,
Jobst
Liebau
,
Lena
Mäler
,
Alexandre
Chenal
,
David
Albesa-Jove
,
Pedro
Merino
,
Marcelo E.
Guerin
Abstract: The phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential peripheral membrane glycosyltransferase that initiates the biosynthetic pathway of phosphatidyl-myo-inositol mannosides (PIMs), key structural elements and virulence factors of Mycobacterium tuberculosis. PimA undergoes functionally important conformational changes, including (i) α-helix-to-β-strand and β-strand-to-α-helix transitions, and (ii) an ‘open-to-closed’ motion between the two Rossmann-fold domains, a conformational change necessary to generate a catalytically competent active site. In previous work, we established that GDP-Man and GDP stabilize the enzyme and facilitate the switch to a more compact active state. To determine the structural contribution of the mannose ring in such activation mechanism we analyzed a series of chemical derivatives, including mannose-phosphate (Man-P) and mannose-pyrophosphate-ribose (Man-PP-RIB), and additional GDP derivatives, as pyrophosphate-ribose (PP-RIB) and GMP, by the combined used of X-ray crystallography, limited proteolysis, circular dichroism, isothermal titration calorimetry and Small Angle X-ray Scattering methods. Although the β-phosphate is present, we found that the mannose ring, neither covalently attached to phosphate (Man-P) nor to PP-RIB (Man-PP-RIB), does promote the switch to the active compact form of the enzyme. Therefore, the nucleotide moiety of GDP-Man, and not the sugar ring, facilitates the ‘open-to-closed’ motion, with the β-phosphate group providing the high affinity binding to PimA. Altogether, the experimental data, contribute to a better understanding of the structural determinants involved in the ‘open-to-closed’ motion observed not only in PimA, but also visualized/predicted in other glycosyltransferases. In addition, the experimental data might prove useful for the discovery/development of PimA and/or glycosyltransferase inhibitors.
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Jul 2020
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[20113]
Open Access
Abstract: The human gut microbiota plays a central role not only in regulating the metabolism of nutrients but also promoting immune homeostasis, immune responses and protection against pathogen colonization. The genome of the Gram-negative symbiont Bacteroides thetaiotaomicron, a dominant member of the human intestinal microbiota, encodes polysaccharide utilization loci PULs, the apparatus required to orchestrate the degradation of a specific glycan. EndoBT-3987 is a key endo-β-N-acetylglucosaminidase (ENGase) that initiates the degradation/processing of mammalian high-mannose-type (HM-type) N-glycans in the intestine. Here, we provide structural snapshots of EndoBT-3987, including the unliganded form, the EndoBT-3987-Man9GlcNAc2Asn substrate complex, and two EndoBT-3987-Man9GlcNAc and EndoBT-3987-Man5GlcNAc product complexes. In combination with alanine scanning mutagenesis and activity measurements we unveil the molecular mechanism of HM-type recognition and specificity for EndoBT-3987 and an important group of the GH18 ENGases, including EndoH, an enzyme extensively used in biotechnology, and for which the mechanism of substrate recognition was largely unknown.
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Feb 2020
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[15304]
Open Access
Abstract: Endoglycosidase S (EndoS) is a bacterial endo-β-N-acetylglucosaminidase that specifically catalyzes the hydrolysis of the β-1,4 linkage between the first two N-acetylglucosamine residues of the biantennary complex-type N-linked glycans of IgG Fc regions. It is used for the chemoenzymatic synthesis of homogeneously glycosylated antibodies with improved therapeutic properties, but the molecular basis for its substrate specificity is unknown. Here, we report the crystal structure of the full-length EndoS in complex with its oligosaccharide G2 product. The glycoside hydrolase domain contains two well-defined asymmetric grooves that accommodate the complex-type N-linked glycan antennae near the active site. Several loops shape the glycan binding site, thereby governing the strict substrate specificity of EndoS. Comparing the arrangement of these loops within EndoS and related endoglycosidases, reveals distinct-binding site architectures that correlate with the respective glycan specificities, providing a basis for the bioengineering of endoglycosidases to tailor the chemoenzymatic synthesis of monoclonal antibodies.
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May 2018
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[15304]
Abstract: Glycolipids play a central role in a variety of important biological processes in all living organisms. PatA is a membrane acyltransferase involved in the biosynthesis of phosphatidyl-myo-inositol mannosides (PIMs), key structural elements and virulence factors of Mycobacterium tuberculosis. PatA catalyzes the transfer of a palmitoyl moiety from palmitoyl-CoA to the 6-position of the mannose ring linked to the 2-position of inositol in PIM1/PIM2. We report here the crystal structure of PatA in the presence of 6-O-palmitoyl-α-D-mannopyranoside, unraveling the acceptor binding mechanism. The acceptor mannose ring localizes in a cavity at the end of a surface-exposed, long groove where the active site is located, whereas the palmitate moiety accommodates into a hydrophobic pocket deeply buried in the α/β core of the protein. Both fatty acyl chains of the PIM2 acceptor are essential for the reaction to take place, highlighting their critical role in the generation of a competent active site. By the use of combined structural and quantum-mechanics/molecular-mechanics (QM/MM) metadynamics we unravel the catalytic mechanism of PatA at the atomic-electronic level. Our study provides a detailed structural rationale for a stepwise reaction, with the generation of a tetrahedral transition state for the rate-determining step. Finally, the crystal structure of PatA in the presence of β-D-mannopyranose and palmitate suggest an inhibitory mechanism for the enzyme, providing exciting possibilities for inhibitor design and the discovery of chemotherapeutic agents against this major human pathogen.
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Nov 2017
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