Krios I-Titan Krios I at Diamond
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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.
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Nov 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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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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David
Albesa-Jove
,
Javier
Romero-Garcia
,
Enea
Sancho-Vaello
,
F.-Xabier
Contreras
,
Ane
Rodrigo-Unzueta
,
Natalia
Comino
,
Ana
Carreras-Gonzalez
,
Pedro
Arrasate
,
Saioa
Urresti
,
Xevi
Biarnes
,
Antoni
Planas
,
Marcelo
Guerin
Diamond Proposal Number(s):
[8302, 10130]
Abstract: Glycosyltransferases (GTs) play a central role in nature. They catalyze the transfer of a sugar moiety to a broad range of acceptor substrates. GTs are highly selective enzymes, allowing the recognition of subtle structural differences in the sequences and stereochemistry of their sugar and acceptor substrates. We report here a series of structural snapshots of the reaction center of the retaining glucosyl-3-phosphoglycerate synthase (GpgS). During this sequence of events, we visualize how the enzyme guides the substrates into the reaction center where the glycosyl transfer reaction takes place, and unveil the mechanism of product release, involving multiple conformational changes not only in the substrates/products but also in the enzyme. The structural data are further complemented by metadynamics free-energy calculations, revealing how the equilibrium of loop conformations is modulated along these itineraries. The information reported here represent an important contribution for the understanding of GT enzymes at the molecular level.
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Jun 2017
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8302, 10130]
Open Access
Abstract: ADP-glucose pyrophosphorylase (AGPase) controls bacterial glycogen and plant starch biosynthetic pathways, the most common carbon storage polysaccharides in nature. AGPase activity is allosterically regulated by a series of metabolites in the energetic flux within the cell. Very recently, we reported the first crystal structures of the paradigmatic AGPase from Escherichia coli (EcAGPase) in complex with its preferred physiological negative and positive allosteric regulators, adenosine-5′-monophosphate (AMP) and fructose-1,6-bisphosphate (FBP), respectively. However, the understanding of the molecular mechanism by which AMP and FBP allosterically modulates EcAGPase enzymatic activity still remains enigmatic. Here we found that single point mutations of key residues in the AMP binding site decrease its inhibitory effect, but also clearly abolish the overall AMP-mediated stabilization effect in wild-type EcAGPase. Single point mutations of key residues for FBP binding did not revert the AMP-mediated stabilization. Strikingly, an EcAGPase·Arg130Ala mutant displayed a dramatic increase in the activity when compared with wild-type EcAGPase, and this increase correlated with a significant increment of glycogen content in vivo. The crystal structure of EcAGPase·Arg130Ala revealed unprecedented conformational changes in structural elements involved in the allosteric signal transmission. Altogether, we propose a model in which the positive and negative energy reporters regulate AGPase catalytic activity via intra- and inter-protomer crosstalk, with a ′sensory motif′ and two loops RL1 and RL2 flanking the ATP binding site playing a significant role. The information reported herein provides exciting possibilities for industrial/biotechnological applications.
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Feb 2017
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[10130, 8302]
Abstract: ADP-glucose pyrophosphorylase (AGPase) catalyzes the rate-limiting step of bacterial glycogen and plant starch biosynthesis, the most common carbon storage polysaccharides in nature. A major challenge is to understand how AGPase activity is regulated by metabolites in the energetic flux within the cell. Here we report crystal structures of the homotetrameric AGPase from Escherichia coli in complex with its physiological positive and negative allosteric regulators, fructose-1,6-bisphosphate (FBP) and AMP, and sucrose in the active site. FBP and AMP bind to partially overlapping sites located in a deep cleft between glycosyltransferase A-like and left-handed β helix domains of neighboring protomers, accounting for the fact that sensitivity to inhibition by AMP is modulated by the concentration of the activator FBP. We propose a model in which the energy reporters regulate EcAGPase catalytic activity by intra-protomer interactions and inter-protomer crosstalk, with a sensory motif and two regulatory loops playing a prominent role.
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Sep 2016
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I04-Macromolecular Crystallography
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David
Albesa-Jove
,
Natalia
Comino
,
Montse
Tersa
,
Elisabeth
Mohorko
,
Saioa
Urresti
,
Elisa
Dainese
,
Laurent R.
Chiarelli
,
Maria Rosalia
Pasca
,
Riccardo
Manganelli
,
Vadim
Makarov
,
Giovanna
Riccardi
,
Dmitri I.
Svergun
,
Rudi
Glockshuber
,
Marcelo
Guerin
Diamond Proposal Number(s):
[8302, 10130]
Open Access
Abstract: Rv2466c is a key oxidoreductase that mediates the reductive activation of TP053, a thienopyrimidine derivative that kills replicating and non-replicating Mycobacterium tuberculosis, but whose mode of action remains enigmatic. Rv2466c is a homodimer in which each subunit displays a modular architecture comprising a canonical thioredoxin fold with a Cys19-Pro20-Trp21-Cys22 motif, and an insertion consisting of a four α-helical bundle and a short α-helical hairpin. Strong evidence is provided for dramatic conformational changes during the Rv2466c redox cycle, which are essential for TP053 activity. Strikingly, a new crystal structure of the reduced form of Rv2466c revealed the binding of a C-terminal extension in α-helical conformation to a pocket next to the active site cysteine pair at the interface between the thioredoxin domain and the helical insertion domain. The ab initio low-resolution envelopes obtained from small angle X-ray scattering showed that the fully reduced form of Rv2466c adopts a ′closed′ compact conformation in solution, similar to that observed in the crystal structure. In contrast, the oxidized form of Rv2466c displays an ′open′ conformation, where tertiary structural changes in the α-helical subdomain suffice to account for the observed conformational transitions. Altogether our structural, biochemical and biophysical data strongly support a model in which the formation of the catalytic disulfide bond upon TP053 reduction triggers local structural changes that open the substrate binding site of Rv2466c allowing the release of the activated, reduced form of TP053. Our studies suggest that similar structural changes might have a functional role in other members of the thioredoxin-fold superfamily.
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Nov 2015
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I04-Macromolecular Crystallography
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David
Giganti
,
David
Albesa-Jove
,
Saioa
Urresti
,
Ane
Rodrigo-Unzueta
,
Mariano A
Martínez
,
Natalia
Comino
,
Nathalie
Barilone
,
Marco
Bellinzoni
,
Alexandre
Chenal
,
Marcelo
Guerin
,
Pedro M.
Alzari
Diamond Proposal Number(s):
[8302, 10130]
Abstract: Secondary structure refolding is a key event in biology as it modulates the conformation of many proteins in the cell, generating functional or aberrant states. The crystal structures of mannosyltransferase PimA reveal an exceptional flexibility of the protein along the catalytic cycle, including β-strand–to–α-helix and α-helix–to–β-strand transitions. These structural changes modulate catalysis and are promoted by interactions of the protein with anionic phospholipids in the membrane.
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Nov 2014
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I02-Macromolecular Crystallography
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David
Albesa-Jove
,
Laurent R.
Chiarelli
,
Vadim
Makarov
,
Maria Rosalia
Pasca
,
Saioa
Urresti
,
Giorgia
Mori
,
Elena
Salina
,
Anthony
Vocat
,
Natalia
Comino
,
Elisabeth
Mohorko
,
Svetlana
Ryabova
,
Bernhard
Pfieiffer
,
Ana Luisa De Jesus
Lopes Ribeiro
,
Ane
Rodrigo-Unzueta
,
Montse
Tersa
,
Giuseppe
Zanoni
,
Silvia
Buroni
,
Karl-Heinz
Altmann
,
Ruben C.
Hartkoorn
,
Rudi
Glockshuber
Diamond Proposal Number(s):
[8302]
Abstract: The emergence of multidrug- and extensively drug-resistant strains of Mycobacterium tuberculosis highlights the need to discover new antitubercular agents. Here we describe the synthesis and characterization of a new series of thienopyrimidine (TP) compounds that kill both replicating and non-replicating M. tuberculosis. The strategy to determine the mechanism of action of these TP derivatives was to generate resistant mutants to the most effective compound TP053 and to isolate the genetic mutation responsible for this phenotype. The only non-synonymous mutation found was a g83c transition in the Rv2466c gene, resulting in the replacement of tryptophan 28 by a serine. The Rv2466c overexpression increased the sensitivity of M. tuberculosis wild-type and resistant mutant strains to TP053, indicating that TP053 is a prodrug activated by Rv2466c. Biochemical studies performed with purified Rv2466c demonstrated that only the reduced form of Rv2466c can activate TP053. The 1.7 angstrom resolution crystal structure of the reduced form of Rv2466c, a protein whose expression is transcriptionally regulated during the oxidative stress response, revealed a unique homodimer in which a beta-strand is swapped between the thioredoxin domains of each subunit. A pronounced groove harboring the unusual active-site motif CPWC might account for the uncommon reactivity profile of the protein. The mutation of Trp28Ser clearly predicts structural defects in the thioredoxin fold, including the destabilization of the dimerization core and the CPWC motif, likely impairing the activity of Rv2466c against TP053. Altogether our experimental data provide insights into the molecular mechanism underlying the anti- mycobacterial activity of TP-based compounds, paving the way for future drug development programmes.
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Jul 2014
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