I24-Microfocus Macromolecular Crystallography
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Sebastian
Perrone
,
Javier O.
Cifuente
,
Leonardo
Mastrella
,
Alberto
Marina
,
Beatriz
Trastoy
,
Julia
Becker-Kettern
,
Jean-François
Conrotte
,
Adrià
Alcaide-Jiménez
,
Francisco
Corzana
,
Enrico
Glaab
,
Marcelo E.
Guerin
,
Carole L.
Linster
Diamond Proposal Number(s):
[28360]
Open Access
Abstract: L-serine is a critical structural constituent of proteins and membrane phospholipids, playing major roles in cell signaling, metabolism and development. L-Serine is synthesized through a conserved de novo pathway starting from the glycolytic intermediate 3-phosphoglycerate (PGA), being oxidized by 3-phosphoglycerate dehydrogenase (PHGDH) into 3-phosphohydroxypyruvate (PHP). In certain organisms, PHGDH operates as a transhydrogenase using α-ketoglutarate rather than NAD+ as the final electron acceptor and producing both PHP and D-2-hydroxyglutarate (2HG). We provide high-resolution X-ray crystal structures of the transhydrogenase Ser33 from Saccharomyces cerevisiae, in complex with the cofactor NADH, and with PGA, PHP, 2HG and the negative allosteric regulator L-serine. Combining extensive alanine scanning mutagenesis, enzyme activity assays and kinetics, molecular dynamics simulations, biophysical methods, and phylogenetic analysis, we establish the molecular basis of substrate recognition, transhydrogenase activity, and allosteric inhibition mechanisms, including the role of an N-terminal extension in the regulation of eukaryotic Type II PHGDHs.
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Jun 2026
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Krios I-Titan Krios I at Diamond
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Valerio
Di Domenico
,
Jorick
Franceus
,
Leonardo
Mastrella
,
Emma
De Beul
,
Adrià
Alcaide-Jiménez
,
Francisco
Paredes-Martinez
,
Juan Carlos
Villegas-Ruiz
,
Elena
Holden
,
Alejandro Delgado
Rey
,
Cecilia
D'Angelo
,
Javier O.
Cifuente
,
Weston B.
Struwe
,
Ricardo M.
Biondi
,
Alberto
Marina
,
Sean R.
Connell
,
Justin L. P.
Benesch
,
Patricia
Casino
,
Christophe
Colleoni
,
Tom
Desmet
,
Marcelo E.
Guerin
Diamond Proposal Number(s):
[17171, 31586]
Open Access
Abstract: Protein phosphorylation is a universal regulatory mechanism, controlling virtually all aspects of bacterial physiology and pathogenesis, yet histidine phosphorylation remains among the least understood. The histidine phosphocarrier protein HPr not only drives bacterial glucose transmembrane uptake through the phosphotransferase system (PTS), but also controls key enzymes for central carbon metabolism, including glycogen phosphorylase (GP). Here we report cryoEM structures of multimeric Escherichia coli EcGP and their complexes with EcHPr. The EM maps reveal an unanticipated density at H806 of EcGP, consistent with histidine phosphorylation within a histidine-rich pocket at the N-terminal domain. Enzymatic assays reveal that EcHPr transfers a phosphoryl group to the N1 position of a histidine residue in EcGP. Through an integrative structural, mutational and functional approach, we uncover the molecular basis of EcHPr- EcGP selectivity and define the allosteric mechanism by which EcHPr regulates EcGP. We establish histidine phosphorylation as a mechanism of GP regulation, expanding the traditional paradigm of glycogen metabolism control in bacteria.
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Apr 2026
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[28394]
Open Access
Abstract: Bacterial interactions are vital for adapting to changing environments, with quorum sensing (QS) systems playing a central role in coordinating behaviors through small signaling molecules. The RRNPPA family is the prevalent QS systems in Bacillota and mediating communication through secreted oligopeptides, which are processed into active pheromones by extracellular proteases. Notably, in several cases the propeptides show the presence of multiple putative pheromones within their sequences, which has been proposed as a mechanism to diversify peptide-receptor specificity and potentially facilitate new functions. However, neither the processes governing the maturation of propeptides containing multiple pheromones, nor their functional significance has been evaluated. Here, using 2 Rap systems from bacteriophages infecting Bacillus subtilis that exhibit different types of pheromone duplication in their propeptides, we investigate the maturation process and the molecular and functional activities of the produced pheromones. Our results reveal that distinct maturation processes generate multiple mature pheromones, which bind to receptors with varying affinities but produce identical structural and biological responses. These findings add additional layers in the complexity of QS communication and regulation, opening new possibilities for microbial social behaviors, highlighting the intricate nature of bacterial interactions and adaptation.
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Aug 2024
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I24-Microfocus Macromolecular Crystallography
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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.
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Jul 2024
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[2452]
Open Access
Abstract: Phages can use a small-molecule communication arbitrium system to coordinate lysis–lysogeny decisions, but the underlying mechanism remains unknown. Here we determined that the arbitrium system in Bacillus subtilis phage phi3T modulates the bacterial toxin–antitoxin system MazE–MazF to regulate the phage life cycle. We show that phi3T expresses AimX and YosL, which bind to and inactivate MazF. AimX also inhibits the function of phi3T_93, a protein that promotes lysogeny by binding to MazE and releasing MazF. Overall, these mutually exclusive interactions promote the lytic cycle of the phage. After several rounds of infection, the phage-encoded AimP peptide accumulates intracellularly and inactivates the phage antiterminator AimR, a process that eliminates aimX expression from the aimP promoter. Therefore, when AimP increases, MazF activity promotes reversion back to lysogeny, since AimX is absent. Altogether, our study reveals the evolutionary strategy used by arbitrium to control lysis–lysogeny by domesticating and fine-tuning a phage-defence mechanism.
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Jan 2024
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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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.
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Jun 2023
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[28394]
Open Access
Abstract: Stl, the master repressor of the Staphylococcus aureus pathogenicity islands (SaPIs), targets phage-encoded proteins to derepress and synchronize the SaPI and the helper phage life cycles. To activate their cycle, some SaPI Stls target both phage dimeric and phage trimeric dUTPases (Duts) as antirepressors, which are structurally unrelated proteins that perform identical functions for the phage. This intimate link between the SaPI’s repressor and the phage inducer has imposed an evolutionary optimization of Stl that allows the interaction with Duts from unrelated organisms. In this work, we structurally characterize this sophisticated mechanism of specialization by solving the structure of the prototypical SaPIbov1 Stl in complex with a prokaryotic and a eukaryotic trimeric Dut. The heterocomplexes with Mycobacterium tuberculosis and Homo sapiens Duts show the molecular strategy of Stl to target trimeric Duts from different kingdoms. Our structural results confirm the participation of the five catalytic motifs of trimeric Duts in Stl binding, including the C-terminal flexible motif V that increases the affinity by embracing Stl. In silico and in vitro analyses with a monomeric Dut support the capacity of Stl to recognize this third family of Duts, confirming this protein as a universal Dut inhibitor in the different kingdoms of life.
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Jan 2023
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[28394]
Open Access
Abstract: Antifungal proteins (AFPs) are promising antimicrobial compounds that represent a feasible alternative to fungicides. Penicillium expansum encodes three phylogenetically distinct AFPs (PeAfpA, PeAfpB and PeAfpC) which show different antifungal profiles and fruit protection effects. To gain knowledge about the structural determinants governing their activity, we solved the crystal structure of PeAfpB and rationally designed five PeAfpA::PeAfpB chimeras (chPeAFPV1-V5). Chimeras showed significant differences in their antifungal activity. chPeAFPV1 and chPeAFPV2 improved the parental PeAfpB potency, and it was very similar to that of PeAfpA. chPeAFPV4 and chPeAFPV5 showed an intermediate profile of activity compared to the parental proteins while chPeAFPV3 was inactive towards most of the fungi tested. Structural analysis of the chimeras evidenced an identical scaffold to PeAfpB, suggesting that the differences in activity are due to the contributions of specific residues and not to induced conformational changes or structural rearrangements. Results suggest that mannoproteins determine protein interaction with the cell wall and its antifungal activity while there is not a direct correlation between binding to membrane phospholipids and activity. This work provides new insights about the relevance of sequence motifs and the feasibility of modifying protein specificity, opening the door to the rational design of chimeras with biotechnological applicability.
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Nov 2022
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[16258]
Open Access
Abstract: Mobile genetic elements control their life cycles by the expression of a master repressor, whose function must be disabled to allow the spread of these elements in nature. Here, we describe an unprecedented repression-derepression mechanism involved in the transfer of Staphylococcus aureus pathogenicity islands (SaPIs). Contrary to the classical phage and SaPI repressors, which are dimers, the SaPI1 repressor StlSaPI1 presents a unique tetrameric conformation never seen before. Importantly, not just one but two tetramers are required for SaPI1 repression, which increases the novelty of the system. To derepress SaPI1, the phage-encoded protein Sri binds to and induces a conformational change in the DNA binding domains of StlSaPI1, preventing the binding of the repressor to its cognate StlSaPI1 sites. Finally, our findings demonstrate that this system is not exclusive to SaPI1 but widespread in nature. Overall, our results characterize a novel repression-induction system involved in the transfer of MGE-encoded virulence factors in nature.
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Oct 2022
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[28394]
Open Access
Abstract: The arbitrium system is employed by phages of the SPbeta family to communicate with their progeny during infection to decide either to follow the lytic or the lysogenic cycle. The system is controlled by a peptide, AimP, that binds to the regulator AimR, inhibiting its DNA-binding activity and expression of aimX. Although the structure of AimR has been elucidated for phages SPβ and phi3T, there is still controversy regarding the molecular mechanism of AimR function, with two different proposed models for SPβ. In this study, we deepen our understanding of the system by solving the structure of an additional AimR that shows chimerical characteristics with the SPβ receptor. The crystal structures of this AimR (apo, AimP-bound and DNA-bound) together with in vitro and in vivo analyses confirm a mechanism of action by AimP-induced conformational restriction, shedding light on peptide specificity and cross regulation with relevant biological implications.
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Jun 2022
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