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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

Ligand-binding site of AccA.Ligands and protein residues involved in the ligand binding are shown as stick. Hydrogen bonds are shown as dashed lines in black (for distances below 3.2 Å) and magenta (for distances between 3.2 and 3.4 Å). (A) agrocinopine A. (B) agrocin 84. (C) L-arabinose-2-phosphate. (D) D-glucose-2-phosphate. (E) Superimposition of the four ligands bound in the ligand binding site of AccA, agrocinopine A (yellow), agrocin 84 (orange), L-arabinose-2-phosphate (green) and D-glucose-2-phosphate (cyan) are shown as stick.
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ppat.1005071.g004: Ligand-binding site of AccA.Ligands and protein residues involved in the ligand binding are shown as stick. Hydrogen bonds are shown as dashed lines in black (for distances below 3.2 Å) and magenta (for distances between 3.2 and 3.4 Å). (A) agrocinopine A. (B) agrocin 84. (C) L-arabinose-2-phosphate. (D) D-glucose-2-phosphate. (E) Superimposition of the four ligands bound in the ligand binding site of AccA, agrocinopine A (yellow), agrocin 84 (orange), L-arabinose-2-phosphate (green) and D-glucose-2-phosphate (cyan) are shown as stick.

Mentions: Both bound ligands, agrocinopine A and agrocin 84, share a deeply buried pyranose (L-arabinose or D-glucose)-2-phosphate-like moiety which superimposes very well in the AccA binding site, and makes numerous and very similar protein contacts (Fig 4). The pyranose-2-phosphate moiety lies on residues 418–421 from the strand β16 of lobe 2 and is surrounded by the N-terminal loop region 52–54 and the side chains of Tyr145, Trp178, Glu504 and Glu510 from lobe 1, that of Asn284 from the hinge region and those of Met372, Tyr375, Tyr376, Thr430, Glu434 from lobe 2. It makes extensive protein hydrogen bonds: 8 for L-arabinose and 10 for D-glucose (Fig 4A and 4B). Remarkably, the OH1 group of the pyranose is anchored by 4 hydrogen bonds involving Asn54 and Glu510 from lobe 1, Asn284 from the hinge region and Ser419 from lobe 2. These four side chains form a rigid template, which also maintains the lobe closure by interacting together two-by-two (S2a Fig). Moreover, in addition to their pyranose interactions, two of these major residues Asn54 and Ser419 also tightly interact with the phosphate/phosphoramidate groups. Two more hydrogen bonds involving the side chains of two tyrosines 375 and 376 retain the phosphate/phosphoramidate oxygens. In contrast, the sucrose moiety of agrocinopine A and the TM84 part of agrocin 84 make only 3 and 2 polar interactions with AccA, respectively. The sucrose moiety and TM84 occupy a different position 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)

Ligand-binding site of AccA.Ligands and protein residues involved in the ligand binding are shown as stick. Hydrogen bonds are shown as dashed lines in black (for distances below 3.2 Å) and magenta (for distances between 3.2 and 3.4 Å). (A) agrocinopine A. (B) agrocin 84. (C) L-arabinose-2-phosphate. (D) D-glucose-2-phosphate. (E) Superimposition of the four ligands bound in the ligand binding site of AccA, agrocinopine A (yellow), agrocin 84 (orange), L-arabinose-2-phosphate (green) and D-glucose-2-phosphate (cyan) are shown as stick.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1005071.g004: Ligand-binding site of AccA.Ligands and protein residues involved in the ligand binding are shown as stick. Hydrogen bonds are shown as dashed lines in black (for distances below 3.2 Å) and magenta (for distances between 3.2 and 3.4 Å). (A) agrocinopine A. (B) agrocin 84. (C) L-arabinose-2-phosphate. (D) D-glucose-2-phosphate. (E) Superimposition of the four ligands bound in the ligand binding site of AccA, agrocinopine A (yellow), agrocin 84 (orange), L-arabinose-2-phosphate (green) and D-glucose-2-phosphate (cyan) are shown as stick.
Mentions: Both bound ligands, agrocinopine A and agrocin 84, share a deeply buried pyranose (L-arabinose or D-glucose)-2-phosphate-like moiety which superimposes very well in the AccA binding site, and makes numerous and very similar protein contacts (Fig 4). The pyranose-2-phosphate moiety lies on residues 418–421 from the strand β16 of lobe 2 and is surrounded by the N-terminal loop region 52–54 and the side chains of Tyr145, Trp178, Glu504 and Glu510 from lobe 1, that of Asn284 from the hinge region and those of Met372, Tyr375, Tyr376, Thr430, Glu434 from lobe 2. It makes extensive protein hydrogen bonds: 8 for L-arabinose and 10 for D-glucose (Fig 4A and 4B). Remarkably, the OH1 group of the pyranose is anchored by 4 hydrogen bonds involving Asn54 and Glu510 from lobe 1, Asn284 from the hinge region and Ser419 from lobe 2. These four side chains form a rigid template, which also maintains the lobe closure by interacting together two-by-two (S2a Fig). Moreover, in addition to their pyranose interactions, two of these major residues Asn54 and Ser419 also tightly interact with the phosphate/phosphoramidate groups. Two more hydrogen bonds involving the side chains of two tyrosines 375 and 376 retain the phosphate/phosphoramidate oxygens. In contrast, the sucrose moiety of agrocinopine A and the TM84 part of agrocin 84 make only 3 and 2 polar interactions with AccA, respectively. The sucrose moiety and TM84 occupy a different position 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