Modelling and mutational evidence identify the substrate binding site and functional elements in APC amino acid transporters

Abstract

The Amino acid-Polyamine-Organocation (APC) superfamily is the main family of amino acid transporters found in all domains of life and one of the largest families of secondary transporters. Here, using a sensitive homology threading approach and modelling we show that the predicted structure of APC members is extremely similar to the crystal structures of several prokaryotic transporters belonging to evolutionary distinct protein families with different substrate specificities. All of these proteins, despite having no primary amino acid sequence similarity, share a similar structural core, consisting of two V-shaped domains of five transmembrane domains each, intertwined in an antiparallel topology. Based on this model, we reviewed available data on functional mutations in bacterial, fungal and mammalian APCs and obtained novel mutational data, which provide compelling evidence that the amino acid binding pocket is located in the vicinity of the unwound part of two broken helices, in a nearly identical position to the structures of similar transporters. Our analysis is fully supported by the evolutionary conservation and specific amino acid substitutions in the proposed substrate binding domains. Furthermore, it allows predictions concerning residues that might be crucial in determining the specificity profile of APC members. Finally, we show that two cytoplasmic loops constitute important functional elements in APCs. Our work along with different kinetic and specificity profiles of APC members in easily manipulated bacterial and fungal model systems could form a unique framework for combining genetic, in-silico and structural studies, for understanding the function of one of the most important transporter families.

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

“…With this strategy we allowed implicitly larger structural movements to occur in the substrate binding regions, than those introduced by the induced fit docking protocol alone. This way we accounted for transporter flexibility, which was necessary (as discussed below) to obtain docking poses that are in agreement with previous studies (23,29,59).…”

“…1). This residue, previously suggested to be part of the proline binding site (23), is found to interact with proline via a hydrogen bond. The second residue, Glu 138 of PrnB (Glu 211 of Put4p), located in TMS3 and also highly conserved in the family, interacts with proline via a salt bridge.…”

“…The PrnB transporter has been used as a model to address structure-function relationships in YATs, by mutational analysis and cysteine scanning mutagenesis (21)(22)(23). Earlier computational studies suggested that APC members possess the so called "5 ϩ 5"-fold (24), commonly found in the crystal struc-tures of several prokaryotic transporters belonging to evolutionary distinct protein families with different substrate specificities (25)(26)(27)(28).…”

“…Earlier computational studies suggested that APC members possess the so called "5 ϩ 5"-fold (24), commonly found in the crystal struc-tures of several prokaryotic transporters belonging to evolutionary distinct protein families with different substrate specificities (25)(26)(27)(28). By using a sensitive homology threading approach, a three-dimensional model of PrnB was previously generated, using as template the crystal structure of the LeuT transporter of Aquifex aeolicus (23). This model predicted that PrnB, as well as other members of the APC superfamilly and the YAT family, share a common structural core (23).…”

“…By using a sensitive homology threading approach, a three-dimensional model of PrnB was previously generated, using as template the crystal structure of the LeuT transporter of Aquifex aeolicus (23). This model predicted that PrnB, as well as other members of the APC superfamilly and the YAT family, share a common structural core (23). This core consists of two intertwined, antiparallel V-shaped repeating units of five transmembrane segments each (TMS1-5 and TMS6 -10) connected by a relatively long loop and is followed by two additional helices (TMS11 and -12).…”