Employing information theoretic measures and mutagenesis to identify residues critical for drug-proton antiport function in Mdr1p of Candida albicans

Abstract

By employing information theoretic measures, this study presents a structure and functional analysis of a multidrug-proton antiporter Mdr1p of Candida albicans. Since CaMdr1p belongs to drug-proton antiporter (DHA1) family of Major Facilitator Superfamily (MFS) of transporters, we contrasted DHA1 (antiporters) with Sugar Porter family (symporters). Cumulative Relative Entropy (CRE) calculated for these two sets of alignments enabled us to selectively identify conserved residues of not only CaMdr1p but for the entire DHA1 family. Based on CRE, the highest scoring thirty positions were selected and predicted to impart functional specificity to CaMdr1p as well as to other drug-proton antiporters. Nineteen positions wherein the CaMdr1p residue matched with the most frequent amino acid at a particular alignment position of DHA1 members were subjected to site-directed mutagenesis and were replaced with either alanine or leucine. All these mutant variants, except one, displayed either complete or selective sensitivity to the tested drugs. The enhanced susceptibility of these mutant variants was corroborated with the simultaneously abrogated efflux of substrates. Taken together, based on scaled CRE between two MFS sub-families, we could accurately predict the functionally relevant residues of CaMdr1p. An extrapolation of these predictions to the entire DHA1 family members as validated from previously published data shows that these residues are functionally critical in other members of the DHA1 family also.