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Modelling, substrate docking and mutational analysis identify residues essential for function and specificity of the major fungal purine transporter AzgA
Author(s) -
Krypotou Emilia,
Lambrinidis George,
Evangelidis Thomas,
Mikros Emmanuel,
Diallinas George
Publication year - 2014
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/mmi.12646
Subject(s) - biology , symporter , transporter , transmembrane domain , docking (animal) , biochemistry , purine , transmembrane protein , binding site , amino acid , computational biology , gene , enzyme , receptor , medicine , nursing
Summary The AzgA purine/H + symporter of A spergillus nidulans is the founding member of a functionally and phylogenetically distinct transporter family present in fungi, bacteria and plants. Here a valid AzgA topological model is built based on the crystal structure of the E scherichia coli uracil transporter UraA , a member of the nucleobase‐ascorbate transporter ( NAT/NCS 2) family. The model consists of 14 transmembrane, mostly α‐helical, segments ( TMSs ) and cytoplasmic N ‐ and C ‐tails. A distinct compact core of 8 TMSs , made of two intertwined inverted repeats ( TMSs 1–4 and 8–11), is topologically distinct from a flexible domain ( TMSs 5–7 and 12–14). A putative substrate binding cavity is visible between the core and the gate domains. Substrate docking, molecular dynamics and mutational analysis identified several residues critical for purine binding and/or transport in TMS 3, TMS 8 and TMS 10. Among these, Asn 131 ( TMS 3), Asp 339 ( TMS 8) and Glu 394 ( TMS 10) are proposed to directly interact with substrates, while Asp 342 ( TMS 8) might be involved in subsequent substrate translocation, through H + binding and symport. Thus, AzgA and other NAT transporters use topologically similar TMSs and amino acid residues for substrate binding and transport, which in turn implies that AzgA ‐like proteins constitute a distant subgroup of the ubiquitous NAT family.