RRC ID 53601
Author Witz S, Panwar P, Schober M, Deppe J, Pasha FA, Lemieux MJ, Möhlmann T.
Title 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.
Journal PLoS One
Abstract 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.
Volume 9(3)
Pages e91343
Published 2014-1-1
DOI 10.1371/journal.pone.0091343
PII PONE-D-13-53036
PMID 24621654
PMC PMC3951388
MeSH Amino Acid Sequence Arabidopsis* Arabidopsis Proteins / chemistry* Arabidopsis Proteins / genetics Arabidopsis Proteins / metabolism* Binding, Competitive Molecular Docking Simulation* Molecular Sequence Data Mutagenesis, Site-Directed* Mutation Nucleobase Transport Proteins / chemistry* Nucleobase Transport Proteins / genetics Nucleobase Transport Proteins / metabolism* Sequence Homology, Amino Acid* Structure-Activity Relationship Substrate Specificity
IF 2.74
Times Cited 17
Prokaryotes E. coli