Modeling, Substrate Docking, and Mutational Analysis Identify Residues Essential for the Function and Specificity of a Eukaryotic Purine-Cytosine NCS1 Transporter

Abstract

Background:The purine-cytosine FcyB transporter is a prototype member of the NCS1 family. Results: Using homology modeling, substrate docking, and rational mutational analysis, we identify residues critical for function and specificity. Conclusion: Important aspects concerning the molecular mechanism and evolution of transporter specificity are revealed. Significance: The first systematic approach on structure-function-specificity relationships in a eukaryotic NCS1 member is shown.

The Aspergillus nidulans Proline Permease as a Model for Understanding the Factors Determining Substrate Binding and Specificity of Fungal Amino Acid Transporters

“…As a result, homology modeling has been used as a reasonable alternative in cases where no x-ray structure is available (23,48,59,61,(67)(68)(69)(70)(71)(72)(73). Using this approach, combined with induced fit docking and a detailed characterization of the specificity profile, we identified 9 residues that are important for PrnB function, Gly 56 Table 5).…”

Heterologous complementation studies reveal the solute transport profiles of a two-member nucleobase cation symporter 1 (NCS1) family in Physcomitrella patens

“…Fig. 1) (Krypotou et al, 2012;Witz et al, 2014). For AtNCS1 the site directed change E227Q abolishes uracil transport , suggesting that this is important for uracil transport in NCS1.…”

“…Site-directed mutagenesis studies focus on residues within the pocket region that appear to interact with the solute or cation in the A. nidulans FCYB and AtNCS1 (PLUTO) transporters (Krypotou et al, 2012. Although some residues are important for substrate binding or transport, particular amino acid residues and associated replacements do not conform to a "one size fits all" scenario.…”

“…Fig. 1) (Weyand et al, 2008;Krypotou et al, 2012;Mourad et al, 2012;Schein et al, 2013;Witz et al, 2014). Molecular model directed mutagenesis and functional analysis of NCS1 including Aspergillus nidulans FCYB (Krypotou et al, 2012) and recently AtNCS1 (also called PLUTO) corroborate such interactions.…”

“…Fig. 1) (Weyand et al, 2008;Mourad et al, 2012;Krypotou et al, 2012;Schein et al, 2013;Witz et al, 2014). A number of the identified amino acids have been the subject of site-directed mutagenesis and functional analysis in the A. nidulans FCYB and AtNCS1 (Suppl.…”