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PLoS One
2017 May 08;125:e0178162. doi: 10.1371/journal.pone.0178162.
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Structural features of PhoX, one of the phosphate-binding proteins from Pho regulon of Xanthomonas citri.
Pegos VR
,
Santos RML
,
Medrano FJ
,
Balan A
.
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In Escherichia coli, the ATP-Binding Cassette transporter for phosphate is encoded by the pstSCAB operon. PstS is the periplasmic component responsible for affinity and specificity of the system and has also been related to a regulatory role and chemotaxis during depletion of phosphate. Xanthomonas citri has two phosphate-binding proteins: PstS and PhoX, which are differentially expressed under phosphate limitation. In this work, we focused on PhoX characterization and comparison with PstS. The PhoX three-dimensional structure was solved in a closed conformation with a phosphate engulfed in the binding site pocket between two domains. Comparison between PhoX and PstS revealed that they originated from gene duplication, but despite their similarities they show significant differences in the region that interacts with the permeases.
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Fig 1. Phylogenetic relationships between the orthologues of PstS and PhoX.Five groups classify the distinct orthologues found in proteobacteria. All the organisms used in this tree as well as their reference codes are described in Table 1. The tree was generated using MEGA 6.0 software and the neighbor-joining algorithm according MM section. The numbers at the nodes indicate the bootstrap percentages of 1000 replicates. Arrows and black dots indicate gene duplication and horizontal gene transfer, respectively. The colors highlight the different groups and subgroups.
Fig 2. Crystal structure of the phosphate-binding protein PhoX from X. citri.(A) Cartoon representation of the three-dimensional structure of PhoX evidencing the α/β folding and the surface (transparent gray) α-helices and loops from domains I and II are shown in blue and cyan, respectively. The β-sheet is colored in yellow. All the secondary structures are labeled. Phosphate ions are shown as red spheres. (B) Side-view of PhoX in surface with the ion buried inside the ligand-binding pocket between both domains. (C) Protein-protein interactions between chains C and E as observed in the crystallographic structure of PhoX, evidencing the set of three phosphates (red spheres) mediating the interaction. (D) Positioning of phosphates 1, 2 and 3 in domain I of chain E, and phosphate 4 inside the ligand-binding pocket. In the box: details of the positive electrostatic potential.
Fig 3. The ligand-binding pocket of X. citri PhoX and its conservation.(A) Detailed view of the PhoX residues (cyan sticks) that interact with the phosphate ion (red stick). Hydrogen bonds are shown as black trace. (B) Structural based amino acid sequence alignment of X. citri PhoX and PstS with several orthologues with solved structures, showing the high level of conservation of the residues that interact with the ion. Numbers are shown according to PhoX structure. (C) Structural superimposition of X. citri PhoX (in black ribbon) and all of the phosphate-binding proteins structures as deposited in PDB. Proteins are shown as ribbons and the phosphate ion from X. citri structure as green spheres. (D) R.m.s.d. values and the aligned residues after the structural superposition of PhoX (314 residues) and each protein is shown in angstroms. PDB codes and 3-letters represent the following proteins: 4GD5_Cpe, Clostridium perfringens PBP, (light gray); 1TWY_Vch, Vibrio cholerae PBP (light blue); 2Z22_Ype, Yersinia pestis PstS (cyan); 1IXH_Eco, E. coli PstS (yellow); 1PC3_Mtu, M. tuberculosis PstS1 (blue); 4LVQ_Mtu, M. tuberculosis PstS3 (magenta), 4EXL_Spn, Streptococcus pneumoniae PstS1 (green), 5I84_Xac, X. citri PhoX (black) and PstS_Xac, X. citri PstS (red).
Fig 4. Comparison and analysis of the regions of PhoX and PstS that interact with the permeases PstCA.(A) Amino acid sequence alignment of RI and RII from PhoX, PstS and E. coli PstS evidencing the high level of conservation (asterisks). Residues in underlined bold are involved with the phosphate binding. (B) The localization of the RI (yellow) and RII (cyan) regions of PhoX and PstS close to the entrance of the ligand pocket shown in surface view and electrostatic potential. Proteins are shown in the same orientation. Electrostatic potential of RI and RII of PhoX and PstS are shown in colored view surface according to the charges calculated in Pymol. Negative: red, positive: blue and neutral: white.
Fig 5. Spectroscopic analysis of X. citri PstS and PhoX in presence and absence of phosphate.Secondary structure prediction of PstS (A) and PhoX (B) was measured by circular dichroism. Traced line: PstS and PhoX in the absence of phosphate; Black line: PstS and PhoX in the absence of phosphate. Gray line: PhoX and PstS incubated with 30 μM of phosphate. The thermal stability of the proteins in absence (black dots) and presence (gray dots) of phosphate was measured at 222 nm for PstS (C) and PhoX (D). The melting temperature (Tm) was calculated and is shown for both conditions.
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