B21-High Throughput SAXS
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Diamond Proposal Number(s):
[21035]
Open Access
Abstract: Apg2, one of the three cytosolic Hsp110 chaperones in humans, supports reactivation of unordered and ordered protein aggregates by Hsc70 (HspA8). Together with DnaJB1, Apg2 serves to nucleate Hsc70 molecules into sites where productive entropic pulling forces can be developed. During aggregate reactivation, Apg2 performs as a specialized nucleotide exchange factor, but the origin of its specialization is poorly defined. Here we report on the role of the distinctive C-terminal extension present in Apg2 and other metazoan homologs. We found that the first part of this Apg2 subdomain with propensity to adopt α-helical structure interacts with the nucleotide binding domain of Hsc70 in a nucleotide-dependent manner, contributing significantly to the stability of the Hsc70:Apg2 complex. Moreover, the second intrinsically disordered segment of Apg2 C-terminal extension plays an important role as a downregulator of nucleotide exchange. An NMR analysis showed that the interaction with Hsc70 nucleotide binding domain modifies the chemical environment of residues located in important functional sites such as the interface between lobe I and II and the nucleotide binding site. Our data indicate that Apg2 C-terminal extension is a fine-tuner of human Hsc70 activity that optimizes the substrate remodeling ability of the chaperone system.
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Sep 2022
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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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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[20113]
Abstract: Recent reports indicate the Type six secretion system exported effector 8 (Tse8) is a cytoactive effector secreted by the Type VI secretion system (T6SS) of the human pathogen Pseudomonas aeruginosa. The T6SS is a nanomachine that assembles inside of the bacteria and injects effectors/toxins into target cells, providing a fitness advantage over competing bacteria and facilitating host colonisation. Here we present the first crystal structure of Tse8, showing it conserves a putative catalytic triad Lys84-transSer162-Ser186 found among homologues. Furthermore, we measure the binding of phenylmethylsulfonyl fluoride (PMSF) to Tse8. Remarkably, we observed that PMSF binding is dependent on the putative catalytic residue Ser186, providing evidences of its nucleophilic reactivity. This work demonstrates that Tse8 belongs to the Amidase Signature (AS) superfamily. Furthermore, it highlights Tse8 similarity to two family members: the Stenotrophomonas maltophilia Peptide Amidase, and the Glutamyl-tRNAGln amidotransferase subunit A from Staphylococcus aureus.
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Oct 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
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Diamond Proposal Number(s):
[15304]
Abstract: Bacillus subtilis PdaC (BsPdaC) is a membrane-bound, multidomain peptidoglycan N-deacetylase acting on N-acetylmuramic (Mur-NAc) residues and conferring lysozyme resistance to modified cell wall peptidoglycans. BsPdaC contains a C-terminal family 4 carbohydrate esterase (CE4) catalytic domain, but unlike other MurNAc deacetylases, BsPdaC also has GlcNAc deacetylase activity on chitooligosaccharides (COSs), characteristic of chitin deacetylases. To uncover the molecular basis of this dual activity, here we determined the X-ray structure of the BsPdaC CE4 domain at 1.54 Å resolution and analyzed its mode of action on COS substrates. We found that the minimal substrate is GlcNAc3 and that activity increases with the degree of glycan polymerization. COS deacetylation kinetics revealed that BsPdaC operates by a multiple-chain mechanism starting at the internal GlcNAc units and leading to deacetylation of all but the reducing-end GlcNAc residues. Interestingly, BsPdaC shares higher sequence similarity with the peptidoglycan GlcNAc deacetylase SpPgdaA than with other MurNAc deacetylases. Therefore, we used ligand-docking simulations to analyze the dual GlcNAc- and MurNAc-binding specificities of BsPdaC and compared them with those of SpPgdA and BsPdaA, representing peptidoglycan deacetylases highly specific for GlcNAc or MurNAc residues, respectively. BsPdaC retains the conserved Asp–His–His metal-binding triad characteristic of CE4 enzymes acting on GlcNAc residues, differing from MurNAc deacetylases that lack the metal-coordinating Asp residue. BsPdaC contains short loops similar to those in SpPgdA, resulting in an open binding cleft that can accommodate polymeric substrates. We propose that PdaC is the first member of a new subclass of peptidoglycan MurNAc deacetylases.
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Nov 2019
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[15304]
Abstract: Protein aggregate reactivation in metazoans is accomplished by the combined activity of Hsp70, Hsp40 and Hsp110 chaperones. Hsp110s support the refolding of aggregated polypeptides acting as specialized nucleotide exchange factors of Hsp70. We have studied how Apg2, one of the three human Hsp110s, regulates the activity of Hsc70 (HspA8), the constitutive Hsp70 in our cells. Apg2 shows a biphasic behavior: at low concentration, it stimulates the ATPase cycle of Hsc70, binding of the chaperone to protein aggregates and the refolding activity of the system, while it inhibits these three processes at high concentration. When the acidic subdomain of Apg2, a characteristic sequence present in the substrate binding domain of all Hsp110s, is deleted, the detrimental effects occur at lower concentration and are more pronounced, which concurs with an increase in the affinity of the Apg2 mutant for Hsc70. Our data support a mechanism in which Apg2 arrests the chaperone cycle through an interaction with Hsc70(ATP) that might lead to premature ATP dissociation before hydrolysis. In this line, the acidic subdomain might serve as a conformational switch to support dissociation of the Hsc70:Apg2 complex.
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Dec 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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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[15304]
Abstract: Glycosyltransferases (GTs) are a key family of enzymes that catalyses the synthesis of glycosidic bonds in all living organisms. The reaction involves the transfer of a glycosyl moiety and can proceed with retention or inversion of the anomeric configuration. To date, the elucidation of the catalytic mechanism of retaining GTs is of great controversy, particularly for those enzymes containing a putative nucleophilic residue in the active site, for which a double-displacement mechanism has been suggested. Here, we report native ternary complexes of the retaining α1,3-Galactosyltransferase (α3GalT) from Bos taurus - containing such a nucleophile in the active site - in a productive mode for catalysis, in the presence of its sugar donor UDP-Gal, the acceptor substrate lactose, and the divalent cation cofactor. This new experimental evidence supports a front-side substrate-assisted SNi-type reaction for α3GalT, and suggests a conserved common catalytic mechanism among retaining GTs.
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Sep 2017
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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):
[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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