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A Pyranose-2-Phosphate Motif Is Responsible for Both Antibiotic Import and Quorum-Sensing Regulation in Agrobacterium tumefaciens.

El Sahili A, Li SZ, Lang J, Virus C, Planamente S, Ahmar M, Guimaraes BG, Aumont-Nicaise M, Vigouroux A, Soulère L, Reader J, Queneau Y, Faure D, Moréra S - PLoS Pathog. (2015)

Bottom Line: This was structurally and functionally confirmed by experiments using four synthetic compounds: agrocinopine 3'-O-benzoate, L-arabinose-2-isopropylphosphate, L-arabinose-2-phosphate and D-glucose-2-phosphate.Our findings shed light on the role of agrocinopine and antibiotic agrocin 84 on quorum-sensing regulation in A. tumefaciens and reveal how the PBP AccA acts as vehicle for the importation of both molecules by means of a key-recognition motif.It also opens future possibilities for the rational design of antibiotic and anti-virulence compounds against A. tumefaciens or other pathogens possessing similar PBPs.

View Article: PubMed Central - PubMed

Affiliation: Institute for Integrative Biology of the Cell (I2BC), Department of Biophysics, Biochemistry and Structural Biology, CNRS CEA University Paris-Sud, Gif-sur-Yvette, France; Institute for Integrative Biology of the Cell (I2BC), Department of Microbiology, CNRS CEA University Paris-Sud, Gif-sur-Yvette, France.

ABSTRACT
Periplasmic binding proteins (PBPs) in association with ABC transporters select and import a wide variety of ligands into bacterial cytoplasm. They can also take up toxic molecules, as observed in the case of the phytopathogen Agrobacterium tumefaciens strain C58. This organism contains a PBP called AccA that mediates the import of the antibiotic agrocin 84, as well as the opine agrocinopine A that acts as both a nutrient and a signalling molecule for the dissemination of virulence genes through quorum-sensing. Here, we characterized the binding mode of AccA using purified agrocin 84 and synthetic agrocinopine A by X-ray crystallography at very high resolution and performed affinity measurements. Structural and affinity analyses revealed that AccA recognizes an uncommon and specific motif, a pyranose-2-phosphate moiety which is present in both imported molecules via the L-arabinopyranose moiety in agrocinopine A and the D-glucopyranose moiety in agrocin 84. We hypothesized that AccA is a gateway allowing the import of any compound possessing a pyranose-2-phosphate motif at one end. This was structurally and functionally confirmed by experiments using four synthetic compounds: agrocinopine 3'-O-benzoate, L-arabinose-2-isopropylphosphate, L-arabinose-2-phosphate and D-glucose-2-phosphate. By combining affinity measurements and in vivo assays, we demonstrated that both L-arabinose-2-phosphate and D-glucose-2-phosphate, which are the AccF mediated degradation products of agrocinopine A and agrocin 84 respectively, interact with the master transcriptional regulator AccR and activate the quorum-sensing signal synthesis and Ti plasmid transfer in A. tumefaciens C58. Our findings shed light on the role of agrocinopine and antibiotic agrocin 84 on quorum-sensing regulation in A. tumefaciens and reveal how the PBP AccA acts as vehicle for the importation of both molecules by means of a key-recognition motif. It also opens future possibilities for the rational design of antibiotic and anti-virulence compounds against A. tumefaciens or other pathogens possessing similar PBPs.

No MeSH data available.


Related in: MedlinePlus

Ribbon representation of AccA in complex with agrocinopine A shown in its annealing Fo-Fc omit map contoured at 4δ.Agrocinopine A is located in the cleft between the lobe 1 (residues 29–280 and 494–521) in slate and the lobe 2 (residues 285–489) in pink and is represented as yellow stick. The short hinge region is shown in red.
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ppat.1005071.g003: Ribbon representation of AccA in complex with agrocinopine A shown in its annealing Fo-Fc omit map contoured at 4δ.Agrocinopine A is located in the cleft between the lobe 1 (residues 29–280 and 494–521) in slate and the lobe 2 (residues 285–489) in pink and is represented as yellow stick. The short hinge region is shown in red.

Mentions: The mature AccA expression plasmid was obtained by cloning the accA gene lacking the first twenty-nine signal sequence residues that serve for localization to bacterial periplasm. Because AccA shares low sequence identity (around 20%) compared to PBPs with known three dimensional structures, we first solved the structure of seleniated AccA in complex with agrocinopine A at 2.65 Å resolution by single-wavelength anomalous dispersion method. A better resolution structure of this complex at 1.9 Å resolution in a different space group using the native protein was determined. The two agrocinopine-complexed structures are very similar, displaying an average root mean square deviation (rmsd) of 0.35 Å over 489 Cα atoms. By the molecular replacement method, we then solved the structure of AccA in complex with agrocin 84 at 2.15 Å resolution (Table 1). The mature AccA structure is a monomer of 493 residues composed of two lobes, each formed by a central β-sheet flanked by α-helices. The biggest lobe (lobe 1) consists of residues 29–280 and 494–521 and the smallest (lobe 2) comprises the residues 285–489. The two lobes are connected by a very short hinge region of 8 residues defining two short segments (Fig 3). AccA fold belongs to the cluster C within the PBP structural classification [1]. Overall, the ligand bound structures are very similar (average rmsd value of 0.33 Å over 490 Cα atoms) adopting a closed conformation. The major difference concerns the region comprising the residues 403–408, which can move up to 1.7 Å to accommodate the ligand in the binding site.


A Pyranose-2-Phosphate Motif Is Responsible for Both Antibiotic Import and Quorum-Sensing Regulation in Agrobacterium tumefaciens.

El Sahili A, Li SZ, Lang J, Virus C, Planamente S, Ahmar M, Guimaraes BG, Aumont-Nicaise M, Vigouroux A, Soulère L, Reader J, Queneau Y, Faure D, Moréra S - PLoS Pathog. (2015)

Ribbon representation of AccA in complex with agrocinopine A shown in its annealing Fo-Fc omit map contoured at 4δ.Agrocinopine A is located in the cleft between the lobe 1 (residues 29–280 and 494–521) in slate and the lobe 2 (residues 285–489) in pink and is represented as yellow stick. The short hinge region is shown in red.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526662&req=5

ppat.1005071.g003: Ribbon representation of AccA in complex with agrocinopine A shown in its annealing Fo-Fc omit map contoured at 4δ.Agrocinopine A is located in the cleft between the lobe 1 (residues 29–280 and 494–521) in slate and the lobe 2 (residues 285–489) in pink and is represented as yellow stick. The short hinge region is shown in red.
Mentions: The mature AccA expression plasmid was obtained by cloning the accA gene lacking the first twenty-nine signal sequence residues that serve for localization to bacterial periplasm. Because AccA shares low sequence identity (around 20%) compared to PBPs with known three dimensional structures, we first solved the structure of seleniated AccA in complex with agrocinopine A at 2.65 Å resolution by single-wavelength anomalous dispersion method. A better resolution structure of this complex at 1.9 Å resolution in a different space group using the native protein was determined. The two agrocinopine-complexed structures are very similar, displaying an average root mean square deviation (rmsd) of 0.35 Å over 489 Cα atoms. By the molecular replacement method, we then solved the structure of AccA in complex with agrocin 84 at 2.15 Å resolution (Table 1). The mature AccA structure is a monomer of 493 residues composed of two lobes, each formed by a central β-sheet flanked by α-helices. The biggest lobe (lobe 1) consists of residues 29–280 and 494–521 and the smallest (lobe 2) comprises the residues 285–489. The two lobes are connected by a very short hinge region of 8 residues defining two short segments (Fig 3). AccA fold belongs to the cluster C within the PBP structural classification [1]. Overall, the ligand bound structures are very similar (average rmsd value of 0.33 Å over 490 Cα atoms) adopting a closed conformation. The major difference concerns the region comprising the residues 403–408, which can move up to 1.7 Å to accommodate the ligand in the binding site.

Bottom Line: This was structurally and functionally confirmed by experiments using four synthetic compounds: agrocinopine 3'-O-benzoate, L-arabinose-2-isopropylphosphate, L-arabinose-2-phosphate and D-glucose-2-phosphate.Our findings shed light on the role of agrocinopine and antibiotic agrocin 84 on quorum-sensing regulation in A. tumefaciens and reveal how the PBP AccA acts as vehicle for the importation of both molecules by means of a key-recognition motif.It also opens future possibilities for the rational design of antibiotic and anti-virulence compounds against A. tumefaciens or other pathogens possessing similar PBPs.

View Article: PubMed Central - PubMed

Affiliation: Institute for Integrative Biology of the Cell (I2BC), Department of Biophysics, Biochemistry and Structural Biology, CNRS CEA University Paris-Sud, Gif-sur-Yvette, France; Institute for Integrative Biology of the Cell (I2BC), Department of Microbiology, CNRS CEA University Paris-Sud, Gif-sur-Yvette, France.

ABSTRACT
Periplasmic binding proteins (PBPs) in association with ABC transporters select and import a wide variety of ligands into bacterial cytoplasm. They can also take up toxic molecules, as observed in the case of the phytopathogen Agrobacterium tumefaciens strain C58. This organism contains a PBP called AccA that mediates the import of the antibiotic agrocin 84, as well as the opine agrocinopine A that acts as both a nutrient and a signalling molecule for the dissemination of virulence genes through quorum-sensing. Here, we characterized the binding mode of AccA using purified agrocin 84 and synthetic agrocinopine A by X-ray crystallography at very high resolution and performed affinity measurements. Structural and affinity analyses revealed that AccA recognizes an uncommon and specific motif, a pyranose-2-phosphate moiety which is present in both imported molecules via the L-arabinopyranose moiety in agrocinopine A and the D-glucopyranose moiety in agrocin 84. We hypothesized that AccA is a gateway allowing the import of any compound possessing a pyranose-2-phosphate motif at one end. This was structurally and functionally confirmed by experiments using four synthetic compounds: agrocinopine 3'-O-benzoate, L-arabinose-2-isopropylphosphate, L-arabinose-2-phosphate and D-glucose-2-phosphate. By combining affinity measurements and in vivo assays, we demonstrated that both L-arabinose-2-phosphate and D-glucose-2-phosphate, which are the AccF mediated degradation products of agrocinopine A and agrocin 84 respectively, interact with the master transcriptional regulator AccR and activate the quorum-sensing signal synthesis and Ti plasmid transfer in A. tumefaciens C58. Our findings shed light on the role of agrocinopine and antibiotic agrocin 84 on quorum-sensing regulation in A. tumefaciens and reveal how the PBP AccA acts as vehicle for the importation of both molecules by means of a key-recognition motif. It also opens future possibilities for the rational design of antibiotic and anti-virulence compounds against A. tumefaciens or other pathogens possessing similar PBPs.

No MeSH data available.


Related in: MedlinePlus