title: The Aspergillus nidulans proline permease as a model for understanding the factors determining substrate binding and specificity of fungal amino acid transporters.
abstract: Amino acid uptake in fungi is mediated by general and specialized members of the yeast amino acid transporter (YAT) family, a branch of the amino acid polyamine organocation (APC) transporter superfamily. PrnB, a highly specific l-proline transporter, only weakly recognizes other Put4p substrates, its Saccharomyces cerevisiae orthologue. Taking advantage of the high sequence similarity between the two transporters, we combined molecular modeling, induced fit docking, genetic, and biochemical approaches to investigate the molecular basis of this difference and identify residues governing substrate binding and specificity. We demonstrate that l-proline is recognized by PrnB via interactions with residues within TMS1 (Gly(56), Thr(57)), TMS3 (Glu(138)), and TMS6 (Phe(248)), which are evolutionary conserved in YATs, whereas specificity is achieved by subtle amino acid substitutions in variable residues. Put4p-mimicking substitutions in TMS3 (S130C), TMS6 (F252L, S253G), TMS8 (W351F), and TMS10 (T414S) broadened the specificity of PrnB, enabling it to recognize more efficiently l-alanine, l-azetidine-2-carboxylic acid, and glycine without significantly affecting the apparent Km for l-proline. S253G and W351F could transport l-alanine, whereas T414S, despite displaying reduced proline uptake, could transport l-alanine and glycine, a phenotype suppressed by the S130C mutation. A combination of all five Put4p-ressembling substitutions resulted in a functional allele that could also transport l-alanine and glycine, displaying a specificity profile impressively similar to that of Put4p. Our results support a model where residues in these positions determine specificity by interacting with the substrates, acting as gating elements, altering the flexibility of the substrate binding core, or affecting conformational changes of the transport cycle.
authors.affiliation: From the Microbial Molecular Genetics Laboratory, Institute of Biosciences and Applications, NCSR "Demokritos," Agia Paraskevi, 15310 Athens and cgournas@bio.demokritos.gr.
authors.forename: Christos
authors.initials: C
authors.lastname: Gournas
authors.affiliation: the School of Pharmacy, University of Athens, Panepistimiopolis, Athens 15771, Greece.
authors.forename: Thomas
authors.initials: T
authors.lastname: Evangelidis
authors.affiliation: From the Microbial Molecular Genetics Laboratory, Institute of Biosciences and Applications, NCSR "Demokritos," Agia Paraskevi, 15310 Athens and.
authors.forename: Alexandros
authors.initials: A
authors.lastname: Athanasopoulos
authors.affiliation: the School of Pharmacy, University of Athens, Panepistimiopolis, Athens 15771, Greece.
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Mikros
authors.affiliation: From the Microbial Molecular Genetics Laboratory, Institute of Biosciences and Applications, NCSR "Demokritos," Agia Paraskevi, 15310 Athens and vicky@bio.demokritos.gr.
authors.forename: Vicky
authors.initials: V
authors.lastname: Sophianopoulou
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D026905:Amino Acid Transport Systems / Q000737:chemistry* / Q000378:metabolism; D026921:Amino Acid Transport Systems, Neutral / Q000737:chemistry* / Q000378:metabolism; D001233:Aspergillus nidulans / Q000201:enzymology*; D018360:Crystallography, X-Ray; D005656:Fungal Proteins / Q000737:chemistry*; D011392:Proline / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D026905:Amino Acid Transport Systems; D026921:Amino Acid Transport Systems, Neutral; D005656:Fungal Proteins; C033082:proline transporter; D011392:Proline
keywords: Amino Acid Transport; Aspergillus; Gating; In-gel Fluorescence; Ligand Docking; Membrane Transporters; Molecular Modeling; Proline; Site-directed Mutagenesis; l-Azetidine-2-carboxylic