Rational mutational analysis of a multidrug MFS transporter CaMdr1p of Candida albicans by employing a membrane environment based computational approach

• Published in: PLoS computational biology Vol. 5; no. 12; p. e1000624
• Main Authors: Kapoor, Khyati, Rehan, Mohd, Kaushiki, Ajeeta, Pasrija, Ritu, Lynn, Andrew M, Prasad, Rajendra
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 01.12.2009; Public Library of Science (PLoS)
• Subjects: Amino Acid Sequence; ATP Binding Cassette Transporter, Subfamily B - chemistry; ATP Binding Cassette Transporter, Subfamily B - genetics; ATP-Binding Cassette Sub-Family B Member 4; Binding Sites; Candida albicans; Candida albicans - metabolism; Cell Membrane - chemistry; Cell Membrane - physiology; Computational Biology/Protein Structure Prediction; Computational Biology/Sequence Motif Analysis; Computer Simulation; DNA Mutational Analysis - methods; Industrial research; Infectious Diseases/Antimicrobials and Drug Resistance; Infectious Diseases/Fungal Infections; Information theory; Models, Biological; Molecular Sequence Data; Protein Binding; Proteins; Saccharomyces cerevisiae; Scholarships & fellowships; Structure-Activity Relationship; Candida albicans; Amino Acid Sequence; Cell Membrane; P-Glycoproteins; DNA Mutational Analysis; Models, Biological; Computer Simulation; Molecular Sequence Data; Protein Binding; Structure-Activity Relationship; Binding Sites
• ISSN: 1553-7358; 1553-734X; 1553-7358
• DOI: 10.1371/journal.pcbi.1000624

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.