Converting the yeast arginine Can1 permease to a lysine permease [Computational Biology]

January 21st, 2014 by Ghaddar, K., Krammer, E.–M., Miha&jnodot;lovic, N., Brohee, S., Andre, B., Prevost, M.

Amino-acid uptake in yeast cells is mediated by about sixteen plasma membrane permeases, most of which belong to the APC (amino acid, polyamine, organocation) transporter family. These proteins display various substrate specificity ranges. For instance, the general amino-acid permease Gap1 transports all amino acids, whereas Can1 and Lyp1 catalyze specific uptake of arginine and lysine, respectively. Though Can1 and Lyp1 have different narrow substrate specificities, they are close homologs. Here we investigated the molecular rules determining the substrate specificity of the H+-driven arginine-specific permease Can1. Using a Can1-Lyp1 sequence alignment as a guideline and a 3D Can1 structural model based on the crystal structure of the bacterial APC-family arginine-agmatine antiporter, we introduced amino acid substitutions liable to alter Can1 substrate specificity. We show that the single substitution T456S results in a Can1 variant transporting lysine in addition to arginine, and that the combined substitutions T456S and S176N convert Can1 to a Lyp1-like permease. Replacement of a highly conserved glutamate in the Can1 binding site leads to variants (E184Q, E184A) incapable of any amino acid transport, pointing to a potential role for this glutamate in H+ coupling. Measurements of the kinetic parameters of arginine and lysine uptake by the wild-type and mutant Can1 permeases, together with docking calculations for each amino acid in their binding site, suggest a model in which residues at positions 176 and 456 confer substrate selectivity at the ligand-binding stage and/or in the course of conformational changes required for transport.