Structure-Function Relationship of a Plant NCS1 Member – Homology Modeling and Mutagenesis Identified Residues Critical for Substrate Specificity of PLUTO, a Nucleobase Transporter from Arabidopsis

<div><p>Plastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from <i>Arabidopsis thaliana</i> is the only known uracil importer at the inner plastidic membrane which represents the permeability barrier of this organelle. We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from <i>Microbacterium liquefaciens</i> and validated by molecular dynamics simulations. Polar side chains of residues Glu-227 and backbones of Val-145, Gly-147 and Thr-425 are proposed to form the binding site for the three PLUTO substrates uracil, adenine and guanine. Mutational analysis and competition studies identified Glu-227 as an important residue for uracil and to a lesser extent for guanine transport. A differential response in substrate transport was apparent with PLUTO double mutants E227Q G147Q and E227Q T425A, both of which most strongly affected adenine transport, and in V145A G147Q, which markedly affected guanine transport. These differences could be explained by docking studies, showing that uracil and guanine exhibit a similar binding mode whereas adenine binds deep into the catalytic pocket of PLUTO. Furthermore, competition studies confirmed these results. The present study defines the molecular determinants for PLUTO substrate binding and demonstrates key differences in structure-function relations between PLUTO and other NCS1 family members.</p></div

Position of functionally relevant residues in the PLUTO structural model.

<p>(A) The PLUTO homology model was built using I-TASSER server based on the open conformation of MHP1 (PDB ID: 2JLN) and its quality was assessed using the PROCHECK program. Important transmembrane segments involved in nucleobase binding are marked in blue (TMs 1a and 1b), green (TM 3), pink (TM 8) and salmon (TMs 6a and 6b). Residues directly involved in substrate binding are V145, G147, E227 and T425 (marked with yellow boxes and shown in the middle). (B) Evolutionary conservation profile of the PLUTO core protein (TM1-TM10) calculated by Consurf server <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091343#pone.0091343-Ashkenazy1" target="_blank">[23]</a> shows a high conservation of the substrate binding pocket. The PLUTO homology model is colored according to the conservation score (based on ClustalW matrix).</p

Competition studies with PLUTO mutants putatively acting as specificity filter.

<p>Direct uptake studies were performed after heterologous expression of PLUTO (black) and PLUTO mutants W223A (white), F341A (spotted) and I226A (striped) in <i>E. coli</i> cells lacking the endogenous uracil transporter uraA. The uptake was measured for uracil (A), guanine (B) and adenine (C) with concentrations of 20 μM and several competitors were added with ten-fold excess (200 μM). The data represent the mean of net uptake rates of at least three independent experiments ± SE. The asterisks indicate significant differences between PLUTO mutants and the control based on Student's t-test (*  = p<0,05; **  = p<0,01; ***  = p<0,005).</p

Substrate competition studies.

<p>Direct uptake studies were performed after heterologous expression of PLUTO in <i>E. coli</i> cells lacking the endogenous uracil transporter uraA. The uptake of uracil (A), guanine (B) and adenine (C) was measured after the incubation with 20 μM substrate for 2 minutes (control). To investigate the effect of the other substrates, they were added to the uptake medium with ten-fold excess (200 μM). The data represent the mean of net uptake rates of at least three independent experiments ± SE. The asterisks indicate significant differences between PLUTO mutants and the control based on Student's t-test (*  = p<0,05; ***  = p<0,005).</p

Alignment of PLUTO with other NCS1-type protein sequences from different organisms and PLUTO homology model.

<p>(A) NCS1 proteins from <i>Arabidopsis thaliana</i> PLUTO (AED90625), <i>Microbacterium liquefaciens</i> MHP1 (2JLN_A) <i>Aspergillus nidulans</i> FCYB (GI: 169798762), and PLUTO homologs from <i>Oryza sativa</i> (Os02g44680), <i>Zea mays</i> (Zm362848), <i>Vitis vinifera</i> (GSVIVT01033705001), <i>Populus trichocarpa</i> (Pt0006S12110), <i>Brachypodium distachyon</i> (Bradi3g51350) were aligned with ClustalW (<a href="http://www.ebi.ac.uk" target="_blank">www.ebi.ac.uk</a>). The residues are shown in white, gray or black color according to their conservation mode (black for highly conserved residues). Residues marked with asterisks were mutated in the course of this work. Residues marked with red boxes are directly involved in PLUTO substrate binding. Blue boxes indicate two Trp residues which are highly conserved among NCS1 proteins and probably exhibit a function in stabilizing the protein-substrate complex with weak pi-stacking interactions. (B) PLUTO homology model with marked TMs. A three-dimensional model of PLUTO was built using I-TASSER server based on structural information of MHP1. The TMs are marked in colors according to the alignment in Figure 1A. The N-terminus of PLUTO was removed for better visualization.</p

Nucleobase transport activity of different PLUTO mutants.

<p>Nucleobase transport activity of different PLUTO mutants.</p

Schematic overview of competition between the different substrates of PLUTO (red) and transport activities of different PLUTO mutants (black).

<p>The presence of uracil leads to a competitive inhibition of guanine transport and vice versa. Furthermore, adenine leads to a competitive inhibition of guanine transport. The mutation E227Q leads to a loss of uracil and guanine transport actitivity (-), whereas the double mutation V145A G147Q only affects guanine transport. In both double mutants E227Q T425A and G147Q E227Q guanine and adenine transport is affected.</p

Docking poses of substrates.

<p>(A) Schematic overview of the docking poses of uracil (green), guanine (red) and adenine (blue). Uracil and guanine exhibit a similar binding mode, whereas adenine binds deep into the catalytic pocket of PLUTO. (B) Docking pose of guanine. (C) Docking pose of uracil. (D) Docking pose of adenine. Glide 5.0 (Schrodinger software) was used for docking purposes and the standard precision mode was selected to dock the substrates into the receptor site.</p