TY - JOUR
T1 - 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
AU - Witz, Sandra
AU - Panwar, Pankaj
AU - Schober, Markus
AU - Deppe, Johannes
AU - Pasha, Farhan Ahmad
AU - Lemieux, M. Joanne
AU - Möhlmann, Torsten
N1 - KAUST Repository Item: Exported on 2020-10-01
PY - 2014/3/12
Y1 - 2014/3/12
N2 - Plastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from Arabidopsis thaliana 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 Microbacterium liquefaciens 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. 2014 Witz et al.
AB - Plastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from Arabidopsis thaliana 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 Microbacterium liquefaciens 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. 2014 Witz et al.
UR - http://hdl.handle.net/10754/325337
UR - https://dx.plos.org/10.1371/journal.pone.0091343
UR - http://www.scopus.com/inward/record.url?scp=84898009362&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0091343
DO - 10.1371/journal.pone.0091343
M3 - Article
C2 - 24621654
SN - 1932-6203
VL - 9
SP - e91343
JO - PLoS ONE
JF - PLoS ONE
IS - 3
ER -