Federica Laddomada

Centre National de la Recherche Scientifique

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Andréa Dessen

Institut de Biologie Structurale

Mayara M. Miyachiro

Institut de Biologie Structurale

Irina Gutsche

Institut de Biologie Structurale

Valentina Pendolino

University College London

Cécile Breyton

Institut de Biologie Structurale

Dominique Mengin‐Lecreulx

Centre National de la Recherche Scientifique

Viviana Job

Institut de Biologie Structurale

Matthew Jessop

Institute of Cancer Research

Andrea Alfieri

University of Pavia

Marina Mapelli

European Institute of Oncology

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Commissariat à l'Énergie Atomique et aux Énergies Alternatives

Centre National de la Recherche Scientifique

Institut de Biologie Structurale

Université Grenoble Alpes

CEA Grenoble

Université Joseph Fourier

Istituti di Ricovero e Cura a Carattere Scientifico

European Institute of Oncology

Unit of Virus Host Cell Interactions

European Molecular Biology Laboratory

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The MurG glycosyltransferase provides an oligomeric scaffold for the cytoplasmic steps of peptidoglycan biosynthesis in the human pathogen Bordetella pertussis

Scientific Reports (2019)

Federica Laddomada Mayara M. Miyachiro Matthew Jessop Delphine Patin Viviana Job

Peptidoglycan is a major component of the bacterial cell wall and thus a major determinant of cell shape. Its biosynthesis is initiated by several sequential reactions catalyzed by cytoplasmic Mur enzymes. Mur ligases (MurC, -D, -E, and -F) are essential for bacteria, metabolize molecules not present in eukaryotes, and are structurally and biochemically tractable. However, although many Mur inhibitors have been developed, few have shown promising antibacterial activity, prompting the hypothesis that within the cytoplasm, Mur enzymes could exist as a complex whose architecture limits access of small molecules to their active sites. This suggestion is supported by the observation that in many bacteria, mur genes are present in a single operon, and pairs of these genes often are fused to generate a single polypeptide. Here, we explored this genetic arrangement in the human pathogen Bordetella pertussis and show that MurE and MurF are expressed as a single, bifunctional protein. EM, small angle X-ray scattering (SAXS), and analytical centrifugation (AUC) revealed that the MurE-MurF fusion displays an elongated, flexible structure that can dimerize. Moreover, MurE-MurF interacted with the peripheral glycosyltransferase MurG, which formed discrete oligomers resembling 4- or 5-armed stars in EM images. The oligomeric structure of MurG may allow it to play a bona fide scaffolding role for a potential Mur complex, facilitating the efficient conveyance of peptidoglycan-building blocks toward the inner membrane leaflet. Our findings shed light on the structural determinants of a peptidoglycan formation complex involving Mur enzymes in bacterial cell wall formation.

Lipid II

Human pathogen

10.1038/s41598-019-40966-z

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Structural Insights into Protein-Protein Interactions Involved in Bacterial Cell Wall Biogenesis

Antibiotics (2016)

Federica Laddomada Mayara M. Miyachiro Andréa Dessen

The bacterial cell wall is essential for survival, and proteins that participate in its biosynthesis have been the targets of antibiotic development efforts for decades. The biosynthesis of its main component, the peptidoglycan, involves the coordinated action of proteins that are involved in multi-member complexes which are essential for cell division (the “divisome”) and/or cell wall elongation (the “elongasome”), in the case of rod-shaped cells. Our knowledge regarding these interactions has greatly benefitted from the visualization of different aspects of the bacterial cell wall and its cytoskeleton by cryoelectron microscopy and tomography, as well as genetic and biochemical screens that have complemented information from high resolution crystal structures of protein complexes involved in divisome or elongasome formation. This review summarizes structural and functional aspects of protein complexes involved in the cytoplasmic and membrane-related steps of peptidoglycan biosynthesis, with a particular focus on protein-protein interactions whereby disruption could lead to the development of novel antibacterial strategies.

10.3390/antibiotics5020014

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Inscuteable and NuMA proteins bind competitively to Leu-Gly-Asn repeat-enriched protein (LGN) during asymmetric cell divisions

Proceedings of the National Academy of Sciences (2011)

Simone Culurgioni Andrea Alfieri Valentina Pendolino Federica Laddomada Marina Mapelli

Coupling of spindle orientation to cellular polarity is a prerequisite for epithelial asymmetric cell divisions. The current view posits that the adaptor Inscuteable (Insc) bridges between Par3 and the spindle tethering machinery assembled on NuMALGNGαi(GDP), thus triggering apico-basal spindle orientation. The crystal structure of the Drosophila ortholog of LGN (known as Pins) in complex with Insc reveals a modular interface contributed by evolutionary conserved residues. The structure also identifies a positively charged patch of LGN binding to an invariant EPE-motif present on both Insc and NuMA. In vitro competition assays indicate that Insc competes with NuMA for LGN binding, displaying a higher affinity, and that it is capable of opening the LGN conformational switch. The finding that Insc and NuMA are mutually exclusive interactors of LGN challenges the established model of force generators assembly, which we revise on the basis of the newly discovered biochemical properties of the intervening components.

10.1073/pnas.1113077108

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Structure et assemblage de complexes des enzymes Mur, essentielles pour la synthèse de la paroi bactérienne

Federica Laddomada

Les enzymes de la famille Mur (MurA-MurG) sont essentielles pour la survie bacterienne, car elles catalysent les etapes cytoplasmiques de la biosynthese du peptidoglycane, la principale composante de la paroi cellulaire. En outre, les Murs metabolisent des molecules qui sont absentes chez les eucaryotes, et ces enzymes sont structurellement et biochimiquement tractables. Cependant, malgre le fait que nombreux inhibiteurs anti-Mur ont ete developpes, un nombre tres reduit de ces molecules ont montre une activite antibacterienne prometteuse, ce qui a incite l'hypothese selon laquelle, dans le cytoplasme bacterien, les enzymes Mur peuvent exister dans un complexe ou les sites actifs sont a proximite, bloquant donc l'acces de petites molecules venant de l'exterieur. Cette hypothese est soutenue par l'observation selon laquelle, dans de nombreux organismes, les genes codant pour les enzymes Mur sont presents dans un seul operon, souvent dans le meme ordre; en outre, souvent des paires de genes sont fusionnees pour generer un seul polypeptide, preconisant la possibilite que des complexes entre ces enzymes pourraient etre formes des qu'ils sont synthetises. Nous avons obtenu les premieres informations structurales et fonctionnelles sur la forme fusionnee MurE-MurF, presente dans le pathogene humain Bordetella pertussis, et nous avons montre qu'elle interagit avec la glycosyltransferase peripherique MurG, ce qui suggere la presence d'un complexe enzymatique ternaire. De facon interessante, nous avons constate que MurG de B. pertussis est capable de s'associer avec elle-meme et de former differentes especes oligomeriques. Cette decouverte pourrait renforcer le role de MurG en tant que proteine agissant comme une plateform capable d'ancrer d'autres enzymes Mur a la face interne de la membrane cytoplasmique bacterienne. Nos resultats pourront egalement etre explores pour comprendre le role potentiel de MurG en tant que regulateur de l'activite des enzymes de synthese du PG. Ces resultats passionnants ouvriront le chemin vers la comprehension du mecanisme d’interaction des enzymes Mur dans le cytoplasme bacterien et pourraient permettre l'emploi eventuel des Murs comme cibles de facto pour developper de nouveaux antibiotiques.

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