Abstract
In the recent years, molecular modeling and substrate docking, coupled with biochemical and genetic analyses have identified the substrate-binding residues of several amino acid transporters of the yeast amino acid transporter (YAT) family. These consist of (a) residues conserved across YATs that interact with the invariable part of amino acid substrates and (b) variable residues that interact with the side chain of the amino acid substrate and thus define specificity. Secondary structure sequence alignments showed that the positions of these residues are conserved across YATs and could thus be used to predict the specificity of YATs. Here, we discuss the potential of combining molecular modeling and structural alignments with intra-species phylogenetic comparisons of transporters, in order to predict the function of uncharacterized members of the family. We additionally define some orphan branches which include transporters with potentially novel, and to be characterized specificities. In addition, we discuss the particular case of the highly specific l-proline transporter, PrnB, of Aspergillus nidulans, whose gene is part of a cluster of genes required for the utilization of proline as a carbon and/or nitrogen source. This clustering correlates with transcriptional regulation of these genes, potentially leading to the efficient coordination of the uptake of externally provided l-Pro via PrnB and its enzymatic degradation in the cell.
Highlights
Amino acids are the building blocks of proteins and most fungi possess the ability to utilize them as nitrogen and/or carbon sources
Paradigmatic examples are amino acid and purine transporters of A. nidulans [1,2,3], the Arg (Can1) and Lys (Lyp1) specific permeases of yeast, as well as the general amino acid permease, Gap1 [4,5,6] (2) the reverse genetics era, where the first sequences of several transporter genes have been cloned, including Can1, Gap1, the histidine (Hip1) and the proline (Put4) permeases of S. cerevisiae [7,8,9,10], as well as the proline-specific transporter, PrnB, and the γ-amino-n-butyrate transporter, GabA of A. nidulans [11,12] (3) the genomics era, where the whole genome sequences of several species, among the first being those of S. cerevisiae and A. nidulans [13,14], provided the amino acid sequences of a plethora of non-characterized transporter proteins
The first crystal structures of membrane proteins, in combination with 3D modeling and extensive site-directed mutagenesis, boosted the study of transporter structure-function relationships. These approaches have led to the characterization of specificity determinants in model fungal amino acid transport systems (fAATs), such as Can1 and PrnB [16,17]
Summary
Amino acids are the building blocks of proteins and most fungi possess the ability to utilize them as nitrogen and/or carbon sources. The first crystal structures of membrane proteins (see below), in combination with 3D modeling and extensive site-directed mutagenesis, boosted the study of transporter structure-function relationships These approaches have led to the characterization of specificity determinants in model fAATs, such as Can and PrnB [16,17]. It was predicted that using hydropathy profile alignments that the APC superfamily would adopt this fold [28] Based on this notion, the first model of a YAT protein, PrnB, was built using 3D modeling [29], providing the first mutational and kinetic evidence that the substrate-binding pocket of YATs is formed by residues in TM1 and TM6. The shift of the transporter back to an outward-facing open conformation completes the transport cycle, and the protein is ready to perform the cycle [35,36]
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