Modelling, substrate docking and mutational analysis identify residues essential for function and specificity of the major fungal purine transporter AzgA.

Abstract

The AzgA purine/H(+) symporter of Aspergillus 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 Escherichia coli uracil transporter UraA, a member of the nucleobase-ascorbate transporter (NAT/NCS2) 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 TMS3, TMS8 and TMS10. Among these, Asn131 (TMS3), Asp339 (TMS8) and Glu394 (TMS10) are proposed to directly interact with substrates, while Asp342 (TMS8) 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.