Rational mutational analysis of a multidrug MFS transporter CaMdr1p of Candida albicans by employing a membrane environment based computational approach
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Updated: Jun 17, 2026
Small-Scale Plasma Membrane Preparation for the Analysis of Candida albicans Cdr1-mGFPHis
Published on: June 13, 2021
Khyati Kapoor1, Mohd Rehan, Ajeeta Kaushiki
1School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
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.
Insights
Identifying critical residues in Candida albicans multidrug transporter CaMdr1p is key to combating azole resistance. Mutating these residues in Saccharomyces cerevisiae increased drug susceptibility, revealing their importance for transporter function.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- CaMdr1p, a multidrug transporter in *Candida albicans*, is implicated in azole resistance.
- Understanding CaMdr1p structure and function is crucial for developing new antifungal therapies.
Purpose of the Study:
- To computationally predict functionally critical residues of CaMdr1p.
- To validate the functional importance of predicted residues through experimental analysis.
Main Methods:
- Utilized a membrane-based computational approach using Modified Relative Entropy (RE(M)) from Multiple Sequence Alignment (MSA).
- Performed site-directed mutational analysis on predicted critical residues in a heterologous host (*Saccharomyces cerevisiae*).
- Assessed drug susceptibility, substrate efflux, protein expression, and surface localization of mutant variants.
Main Results:
- Mutational analysis of high RE(M) residues in CaMdr1p led to increased drug susceptibility in *S. cerevisiae*.
- Hypersensitivity was linked to abrogated substrate efflux, not altered protein expression or localization.
- A 3D model suggested critical residues maintain inter-helical interactions essential for transporter function.
Conclusions:
- Identified functionally critical residues of CaMdr1p using a computational approach.
- Experimental validation confirmed the role of these residues in CaMdr1p function and drug efflux.
- Findings have implications for understanding and targeting multidrug transporters in other membrane protein families.
