I24-Microfocus Macromolecular Crystallography
|
Christa
Litschko
,
Valerio
Di Domenico
,
Julia
Schultz
,
Sizhe
Li
,
Olga G.
Ovchinnikova
,
Thijs
Voskuilen
,
Andrea
Bethe
,
Javier O.
Cifuente
,
Alberto
Marina
,
Insa
Budde
,
Tim A.
Mast
,
Małgorzata
Sulewska
,
Monika
Berger
,
Falk F. R.
Buettner
,
Todd L.
Lowary
,
Chris
Whitfield
,
Jeroen D. C.
Codée
,
Mario
Schubert
,
Marcelo E.
Guerin
,
Timm
Fiebig
Diamond Proposal Number(s):
[28360]
Open Access
Abstract: Capsules are long-chain carbohydrate polymers that envelop the surfaces of many bacteria, protecting them from host immune responses. Capsule biosynthesis enzymes are potential drug targets and valuable biotechnological tools for generating vaccine antigens. Despite their importance, it remains unknown how structurally variable capsule polymers of Gram-negative pathogens are linked to the conserved glycolipid anchoring these virulence factors to the bacterial membrane. Using Actinobacillus pleuropneumoniae as an example, we demonstrate that CpsA and CpsC generate a poly(glycerol-3-phosphate) linker to connect the glycolipid with capsules containing poly(galactosylglycerol-phosphate) backbones. We reconstruct the entire capsule biosynthesis pathway in A. pleuropneumoniae serotypes 3 and 7, solve the X-ray crystal structure of the capsule polymerase CpsD, identify its tetratricopeptide repeat domain as essential for elongating poly(glycerol-3-phosphate) and show that CpsA and CpsC stimulate CpsD to produce longer polymers. We identify the CpsA and CpsC product as a wall teichoic acid homolog, demonstrating similarity between the biosynthesis of Gram-positive wall teichoic acid and Gram-negative capsules.
|
Jul 2024
|
|
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Javier O.
Cifuente
,
Julia
Schulze
,
Andrea
Bethe
,
Valerio
Di Domenico
,
Christa
Litschko
,
Insa
Budde
,
Lukas
Eidenberger
,
Hauke
Thiesler
,
Isabel
Ramón Roth
,
Monika
Berger
,
Heike
Claus
,
Cecilia
D'Angelo
,
Alberto
Marina
,
Rita
Gerardy-Schahn
,
Mario
Schubert
,
Marcelo E.
Guerin
,
Timm
Fiebig
Diamond Proposal Number(s):
[28360]
Open Access
Abstract: Bacterial capsules have critical roles in host-pathogen interactions. They provide a protective envelope against host recognition, leading to immune evasion and bacterial survival. Here we define the capsule biosynthesis pathway of Haemophilus influenzae serotype b (Hib), a Gram-negative bacterium that causes severe infections in infants and children. Reconstitution of this pathway enabled the fermentation-free production of Hib vaccine antigens starting from widely available precursors and detailed characterization of the enzymatic machinery. The X-ray crystal structure of the capsule polymerase Bcs3 reveals a multi-enzyme machine adopting a basket-like shape that creates a protected environment for the synthesis of the complex Hib polymer. This architecture is commonly exploited for surface glycan synthesis by both Gram-negative and Gram-positive pathogens. Supported by biochemical studies and comprehensive 2D nuclear magnetic resonance, our data explain how the ribofuranosyltransferase CriT, the phosphatase CrpP, the ribitol-phosphate transferase CroT and a polymer-binding domain function as a unique multi-enzyme assembly.
|
Jun 2023
|
|
B21-High Throughput SAXS
|
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.
|
Mar 2023
|
|
I24-Microfocus Macromolecular Crystallography
|
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.
|
Mar 2023
|
|
B21-High Throughput SAXS
I24-Microfocus Macromolecular Crystallography
|
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.
|
Mar 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
|
|
I24-Microfocus Macromolecular Crystallography
|
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.
|
Jul 2021
|
|
Krios I-Titan Krios I at Diamond
|
Diamond Proposal Number(s):
[6916, 17171]
Open Access
Abstract: Glycogen and starch are the major carbon and energy reserve polysaccharides in nature, providing living organisms with a survival advantage. The evolution of the enzymatic machinery responsible for the biosynthesis and degradation of such polysaccharides, led the development of mechanisms to control the assembly and disassembly rate, to store and recover glucose according to cell energy demands. The tetrameric enzyme ADP-glucose pyrophosphorylase (AGPase) catalyzes and regulates the initial step in the biosynthesis of both α-polyglucans. AGPase displays cooperativity and allosteric regulation by sensing metabolites from the cell energy flux. The understanding of the allosteric signal transduction mechanisms in AGPase arises as a long-standing challenge. In this work, we disclose the cryoEM structures of the paradigmatic homotetrameric AGPase from Escherichia coli (EcAGPase), in complex with either positive or negative physiological allosteric regulators, fructose-1,6-bisphosphate (FBP) and AMP respectively, both at 3.0 Å resolution. Strikingly, the structures reveal that FBP binds deeply into the allosteric cleft and overlaps the AMP site. As a consequence, FBP promotes a concerted conformational switch of a regulatory loop, RL2, from a “locked” to a “free” state, modulating ATP binding and activating the enzyme. This notion is strongly supported by our complementary biophysical and bioinformatics evidence, and a careful analysis of vast enzyme kinetics data on single-point mutants of EcAGPase. The cryoEM structures uncover the residue interaction networks (RIN) between the allosteric and the catalytic components of the enzyme, providing unique details on how the signaling information is transmitted across the tetramer, from which cooperativity emerges. Altogether, the conformational states visualized by cryoEM reveal the regulatory mechanism of EcAGPase, laying the foundations to understand the allosteric control of bacterial glycogen biosynthesis at the molecular level of detail.
|
Nov 2020
|
|
I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
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.
|
Sep 2020
|
|
B21-High Throughput SAXS
|
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.
|
Jul 2020
|
|
