Abstract
CaMdr1p is a multidrug MFS transporter of pathogenic
Candida albicans
. An over-expression of the gene encoding this protein is linked to clinically encountered azole resistance. In-depth knowledge of the structure and function of CaMdr1p is necessary for an effective design of modulators or inhibitors of this efflux transporter. Towards this goal, in this study, we have employed a membrane environment based computational approach to predict the functionally critical residues of CaMdr1p. For this, information theoretic scores which are variants of Relative Entropy (Modified Relative Entropy RE
M
) were calculated from Multiple Sequence Alignment (MSA) by separately considering distinct physico-chemical properties of transmembrane (TM) and inter-TM regions. The residues of CaMdr1p with high RE
M
which were predicted to be significantly important were subjected to site-directed mutational analysis. Interestingly, heterologous host
Saccharomyces cerevisiae
, over-expressing these mutant variants of CaMdr1p wherein these high RE
M
residues were replaced by either alanine or leucine, demonstrated increased susceptibility to tested drugs. The hypersensitivity to drugs was supported by abrogated substrate efflux mediated by mutant variant proteins and was not attributed to their poor expression or surface localization. Additionally, by employing a distance plot from a 3D deduced model of CaMdr1p, we could also predict the role of these functionally critical residues in maintaining apparent inter-helical interactions to provide the desired fold for the proper functioning of CaMdr1p. Residues predicted to be critical for function across the family were also found to be vital from other previously published studies, implying its wider application to other membrane protein families.
Membrane proteins belonging to the Major Facilitator Superfamily (MFS) transport molecules, including drugs, across the membrane and are known to be associated with drug resistance. CaMdr1p is one such MFS major multidrug efflux pump whose over-expression is linked to frequently encountered azole resistance in hospital isolates of
C. albicans
. Amino acid residues critical for a protein's function are conserved across members of the protein family. However, the traditional measure of conservation is not a useful parameter in mapping a functionally important residue in membrane proteins e.g., hydrophobically conserved stretches form helical transmembrane regions of the protein and are responsible for membrane localization, which individually have limited effect on binding and transport. We have developed a method that uses information theory to score the conservation of a residue relative to its context within the membrane and hypothesize that these residues would be critical for the protein's function. The relevance of predicted residues in the functioning of MFS is validated on CaMdr1p.