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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

Comparison of microcalorimetry derived enthalpy (ΔH, deep grey), entropic contribution (TΔS, grey) and free binding enthalpy (ΔG, light grey) at 293°K for agrocinopine A, agrocin 84, agrocinopine 3’-O-benzoate, L-arabinose-2-isopropylphosphate, L-arabinose-2-phosphate and D-glucose-2-phosphate.
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ppat.1005071.g005: Comparison of microcalorimetry derived enthalpy (ΔH, deep grey), entropic contribution (TΔS, grey) and free binding enthalpy (ΔG, light grey) at 293°K for agrocinopine A, agrocin 84, agrocinopine 3’-O-benzoate, L-arabinose-2-isopropylphosphate, L-arabinose-2-phosphate and D-glucose-2-phosphate.

Mentions: Ligand binding to the protein AccA was investigated using tryptophan fluorescence spectroscopy, a method exploiting significant conformational changes accompanying the binding. The autofluorescence intensity enhancement correlated with the ligand concentrations between 0.3 and 20 μM and saturated above 50 μM. Titration experiments yielded apparent KD values of 1.3 ± 0.17 μM, 5.88 ± 1.6 μM, 2.93 ± 0.66 μM, 4.79 ± 0.6 μM and 2.5 ± 0.5 μM with agrocinopine A, agrocinopine 3’-O-benzoate, L-arabinose-2-phosphate, L-arabinose-2-isopropylphosphate and D-glucose-2-phosphate, respectively (S4 Fig and S1 Table). No fluorescence intensity change was detected by incubating AccA with L-arabinose, D-glucose or phosphate alone. As expected from models based on the X-ray liganded AccA structures, no autofluorescence signal changes were measured by incubating AccA with glucose-1-phosphate or glucose-6-phosphate, which both are common metabolites in all living organisms. Unexpectedly, no signal was measured with agrocin 84, likely due to the presence of the adenosine moiety of agrocin 84 which provokes a quenching signal. Therefore, we used isothermal titration microcalorimetry to assess the binding of agrocin 84 to AccA, which yielded a mean KD of 1.5 ± 0.41 μM. The mean KD values were 0.3 ± 0.03 μM, 7.5 ± 2.2 μM, 1.33 ± 0.12 μM, 2.2 ± 0.58 μM and 1.16 ± 0.22 μM for agrocinopine A, agrocinopine 3’-O-benzoate, L-arabinose-2-phosphate, L-arabinose-2-isopropylphosphate and D-glucose-2-phosphate respectively, consistent with the values obtained from fluorescence spectroscopy. The isothermal titration microcalorimetry data also confirmed the 1:1 binding stoichiometry and demonstrate a negative enthalpy change upon each ligand binding (S5 Fig and S2 Table), suggesting that the binding is enthalpy driven. The similar binding isotherms for all ligands suggest a same binding mechanism involving polar interactions, in agreement with what is observed in the complexed structures (Fig 5). Nevertheless, the benzoate group of the agrocinopine 3’-O-benzoate molecule appears to be responsible for an entropic effect leading to a 25-fold lower affinity of this ligand with AccA in comparison with agrocinopine A. We could not detect any binding of L-arabinose, D-glucose, nor adenosine monophosphate in line with the results obtained by fluorimetry.


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)

Comparison of microcalorimetry derived enthalpy (ΔH, deep grey), entropic contribution (TΔS, grey) and free binding enthalpy (ΔG, light grey) at 293°K for agrocinopine A, agrocin 84, agrocinopine 3’-O-benzoate, L-arabinose-2-isopropylphosphate, L-arabinose-2-phosphate and D-glucose-2-phosphate.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1005071.g005: Comparison of microcalorimetry derived enthalpy (ΔH, deep grey), entropic contribution (TΔS, grey) and free binding enthalpy (ΔG, light grey) at 293°K for agrocinopine A, agrocin 84, agrocinopine 3’-O-benzoate, L-arabinose-2-isopropylphosphate, L-arabinose-2-phosphate and D-glucose-2-phosphate.
Mentions: Ligand binding to the protein AccA was investigated using tryptophan fluorescence spectroscopy, a method exploiting significant conformational changes accompanying the binding. The autofluorescence intensity enhancement correlated with the ligand concentrations between 0.3 and 20 μM and saturated above 50 μM. Titration experiments yielded apparent KD values of 1.3 ± 0.17 μM, 5.88 ± 1.6 μM, 2.93 ± 0.66 μM, 4.79 ± 0.6 μM and 2.5 ± 0.5 μM with agrocinopine A, agrocinopine 3’-O-benzoate, L-arabinose-2-phosphate, L-arabinose-2-isopropylphosphate and D-glucose-2-phosphate, respectively (S4 Fig and S1 Table). No fluorescence intensity change was detected by incubating AccA with L-arabinose, D-glucose or phosphate alone. As expected from models based on the X-ray liganded AccA structures, no autofluorescence signal changes were measured by incubating AccA with glucose-1-phosphate or glucose-6-phosphate, which both are common metabolites in all living organisms. Unexpectedly, no signal was measured with agrocin 84, likely due to the presence of the adenosine moiety of agrocin 84 which provokes a quenching signal. Therefore, we used isothermal titration microcalorimetry to assess the binding of agrocin 84 to AccA, which yielded a mean KD of 1.5 ± 0.41 μM. The mean KD values were 0.3 ± 0.03 μM, 7.5 ± 2.2 μM, 1.33 ± 0.12 μM, 2.2 ± 0.58 μM and 1.16 ± 0.22 μM for agrocinopine A, agrocinopine 3’-O-benzoate, L-arabinose-2-phosphate, L-arabinose-2-isopropylphosphate and D-glucose-2-phosphate respectively, consistent with the values obtained from fluorescence spectroscopy. The isothermal titration microcalorimetry data also confirmed the 1:1 binding stoichiometry and demonstrate a negative enthalpy change upon each ligand binding (S5 Fig and S2 Table), suggesting that the binding is enthalpy driven. The similar binding isotherms for all ligands suggest a same binding mechanism involving polar interactions, in agreement with what is observed in the complexed structures (Fig 5). Nevertheless, the benzoate group of the agrocinopine 3’-O-benzoate molecule appears to be responsible for an entropic effect leading to a 25-fold lower affinity of this ligand with AccA in comparison with agrocinopine A. We could not detect any binding of L-arabinose, D-glucose, nor adenosine monophosphate in line with the results obtained by fluorimetry.

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