Rational Mutational Analysis of a Multidrug MFS Transporter CaMdr1p of Candida albicans by Employing a Membrane Environment Based Computational Approach

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 REM) 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 REM 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 REM 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.

Insight into Pleiotropic Drug Resistance ATP-binding Cassette Pump Drug Transport through Mutagenesis of Cdr1p Transmembrane Domains*

“…S2). Although such conservation could contribute to broad functions such as the hydrophobic residues required for membrane localization (52), the present study suggests neither variation nor conservation within TMSs dominates the substrate promiscuity of multidrug transporters.…”

Employing Information Theoretic Measures and Mutagenesis to Identify Residues Critical for Drug-Proton Antiport Function in Mdr1p of Candida albicans

“…CRE S calculations also assign a low score to residues which are identically conserved in both these families. Such residues are shown to be MFS-wide-function-specific in our previous study [8]. Thus, we hypothesize that residues having high CRE S score are critical for drug-proton antiport function while residues with low CRE S scores are not expected to be critical for drug-proton antiport function but may be important for family-wide function.…”

“…All the three compounds have been shown to be the substrates of CaMdr1p [8], [17]. The intracellular concentration of these substrates was determined with respect to time.…”

“…As an exception, our criteria of CRE S also picked up two residues D235 and F277, which were earlier reported to be family-wide function-specific [8]. This could probably be because our program does not discriminate between the nature of amino acid present in DHA1 and SP families at a given alignment position.…”

Candida Efflux ATPases and Antiporters in Clinical Drug Resistance

“…This led to computational based analysis of CaMdr1p where membrane environment based approach was employed to rationally predict the critical residues of the protein (Kapoort et al 2009(Kapoort et al , 2010. When rationally selected important residues were subjected to sitedirected mutational analysis followed by phenotyping, the data proved that the predicted residues were indeed important in maintaining inter-helical interactions and function of the transporter (Kapoor et al 2009). The study could show that by employing similar rational approach, critical residues in other transporters can also be predicted (Kapoor et al 2010).…”