Biophysical characterization of an essential protein complex for bacterial cell wall formation

Orientador: Andréa Dessen de Souza e SilvaTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: As interações entre proteínas desempenham papéis biológicos importantes nas células, como no caso da biossíntese do peptideoglicano (PG), maior componente da parede celular bacteriana e responsável por definir a morfologia e proteger a célula do estresse causado pela alta pressão osmótica intracelular presente tanto em bactérias Gram-negativas como Gram-positivas. A síntese do PG é um processo dinâmico e altamente complexo, consistente de muitos passos de síntese que são orquestrados por diferentes enzimas localizadas em diferentes espaços na célula. As ligases Mur (MurC a MurF) são as proteínas responsáveis por catalisar a síntese dos precursores de PG no citoplasma juntamente com as proteínas MurG e MraY, as quais também estão relacionadas com a síntese dos precursores do PG na membrana, lipídeo I e lipídeo II, respectivamente. Esse grupo de proteínas são essenciais para a sobrevivência da bactéria e são encontradas somente em células de organismos procariota, deste modo, tornando-se alvos potenciais para o desenvolvimento de novos antibióticos. No presente trabalho foi realizada a expressão e purificação de MraY de Streptococcus pneumoniae, proteína integral de membrana, e foi analisada a sua possível interação com a enzima MurF. Existem muitas evidências sobre a formação de complexos entre essas proteínas, no entanto este trabalho descreve de forma inédita a interação direta entre as ligases Mur de Streptococcus pneumoniae, as quais formam um mega-complexo em solução. Também observamos que a interação entre essas proteínas está fortemente relacionada com a presença dos seus estados oligoméricos. Outro achado inédito é a habilidade de MurG de se auto associar em diferentes espécies, dependendo da concentração de detergente em solução, representando um processo dinâmico, importante para a montagem do mega-complexo entre as ligases Mur, uma vez que a interação entre essas proteínas também foi observada neste estudo. Assim, a formação do mega-complexo pode ser explorada como um mecanismo regulatório espacial e temporal da síntese do peptideoglicanoAbstract: Protein interactions play important biological roles for cells, as in the case of the biosynthesis of peptidoglycan (PG), the major component of the bacterial cell wall. The PG is involved in the determination of cell morphology and protection against osmotic stress, both in Gram-negative and Gram-positive bacteria. The synthesis of PG is a dynamic and highly complex process, which requires many steps mediated by different enzymes located in different compartments of the cell. The Mur (MurC to MurF) ligases are responsible for the catalysis of the synthesis of PG precursors in the cytoplasm, together with the MurG and MraY proteins, which are related to the synthesis of lipid I and lipid II respectively. These proteins are essential for bacterial survival and are found only in prokaryotic organisms, and are thus potential targets for the development of new antibiotics. Here we present the expression and purification of MraY from Streptococcus pneumoniae, an integral membrane protein, as well as its analysis of the interaction with MurF. There is vast evidence regarding the formation of complexes between these proteins; however, this work describes, for the first time, the direct interaction between the Mur ligases of Streptococcus pneumoniae, which form a mega-complex in solution. We also observed that the interaction between these proteins is strongly related to the presence of their oligomeric forms. Interestingly, we have found that S. pneumoniae MurG is able to self-associate and form different oligomeric species in solution. This fact represents a dynamic process, important for the assembly of a mega-complex within the cytoplasm, and could be representative of a spatial and temporal PG-regulatory mechanismDoutoradoGenetica de MicroorganismosDoutora em Genética e Biologia Molecular2013/02451-0FAPES

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

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 formation9FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP11/52067-6; 2017/12436-9; 2013/02451-0FRISBI [ANR-10-INSB-05-02]; GRAL within the Grenoble Partnership for Structural Biology (PSB) [ANR-10-LABX-49-01]; Rhone-Alpes RegionRegion Auvergne-Rhone-Alpes; Fondation pour la Recherche Medicale (FRM)Fondation pour la Recherche Medicale; fonds FEDER; Centre National de la Recherche Scientifique (CNRS)Centre National de la Recherche Scientifique (CNRS); Commissariat a l'Energie Atomique et aux Energies Alternatives (CEA)French Atomic Energy Commission; University of Grenoble Alpes; EMBL; GIS-Infrastructures en Biologie Sante et Agronomie (IBISA); Laboratoire International Associe BACWALL (CNRS); FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [11/52067-6, 2017/12436-9]; Agence Nationale de la RechercheFrench National Research Agency (ANR) [ANR-13-BSV8-0015-01]; ANRFrench National Research Agency (ANR); Fondation pour la Recherche Medicale (FRM)Fondation pour la Recherche Medicale [FDT20160435484]; FAPESPFundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2013/02451-0

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

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

Architecture and genomic arrangement of the MurE–MurF bacterial cell wall biosynthesis complex

Peptidoglycan (PG) is a central component of the bacterial cell wall, and the disruption of its biosynthetic pathway has been a successful antibacterial strategy for decades. PG biosynthesis is initiated in the cytoplasm through sequential reactions catalyzed by Mur enzymes that have been suggested to associate into a multimembered complex. This idea is supported by the observation that in many eubacteria, mur genes are present in a single operon within the well conserved dcw cluster, and in some cases, pairs of mur genes are fused to encode a single, chimeric polypeptide. We performed a vast genomic analysis using >140 bacterial genomes and mapped Mur chimeras in numerous phyla, with Proteobacteria carrying the highest number. MurE–MurF, the most prevalent chimera, exists in forms that are either directly associated or separated by a linker. The crystal structure of the MurE–MurF chimera from Bordetella pertussis reveals a head-to-tail, elongated architecture supported by an interconnecting hydrophobic patch that stabilizes the positions of the two proteins. Fluorescence polarization assays reveal that MurE–MurF interacts with other Mur ligases via its central domains with K D s in the high nanomolar range, backing the existence of a Mur complex in the cytoplasm. These data support the idea of stronger evolutionary constraints on gene order when encoded proteins are intended for association, establish a link between Mur ligase interaction, complex assembly and genome evolution, and shed light on regulatory mechanisms of protein expression and stability in pathways of critical importance for bacterial survival

Complex Formation between Mur Enzymes from Streptococcus pneumoniae

International audiencePeptidoglycan is one of the major components of the bacterial cell wall, being responsible for shape and stability. Due to its essential nature, its biosynthetic pathway is the target for major antibiotics, and proteins involved in its biosynthesis continue to be targeted for inhibitor studies. The biosynthesis of its major building block, Lipid II, is initiated in the bacterial cytoplasm with the sequential reactions catalyzed by Mur enzymes, which have been suggested to form a multiprotein complex to facilitate shuttling of the building blocks toward the inner membrane. In this work, we purified MurC, MurD, MurE, MurF, and MurG from the human pathogen Streptococcus pneumoniae and characterized their interactions using chemical cross-linking, mass spectrometry, analytical ultracentrifugation, and microscale thermophoresis. Mur ligases interact strongly as binary complexes, with interaction regions mapping mostly to loop regions. Interestingly, MurC, MurD, and MurE display 10-fold higher affinity for each other than for MurF and MurG, suggesting that Mur ligases that catalyze the initial reactions in the peptidoglycan biosynthesis pathway could form a subcomplex that could be important to facilitate Lipid II biosynthesis. The interface between Mur proteins could represent a yet unexplored target for new inhibitor studies that could lead to the development of novel antimicrobials