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 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.

Figure 1. A portion of Multiple Alignment showing conservation and RE_M for each column.

Figure showing a representative portion of the alignment of MFS sequences and is generated using Alscript . The alignment is coloured in a gradient from red to yellow on the basis of decreasing conservation score. Conservation was calculated using a method by Livingstone et al. and the scores are shown as a histogram. The histogram compares the conservation, RE and the RE_M scores for the alignment columns shows in the figure. Results of selected mutations in CaMdr1p are indicated by triangles, green being sensitive while red are resistant. RE_M scores are better indicators of functional relevance than physicochemical conservation.

Figure 2. Distribution curve of RE_M values of complete MSA.

Panel A: Histogram of the RE_M scores (+ve/background frequency) vs frequency for all positions of MSA of the MFS transporters. The top 30 RE_M positions depicted in the boxed region were selected for further analysis. The inset represents the same data by a line graph. B: The table shows 16 out of the top 30 high RE_M alignment positions where the residue in CaMdr1p matched with the most frequent amino acid at that particular position in a MSA of 342 MFS members. Their predicted location with respect to CaMdr1p is also displayed in the next column.

Figure 3. Drug susceptibility and transport assays of mutant variants of CaMDR1-GFP in S. cerevisiae.

Panel A: Drug resistance profile of wild type and mutant CaMDR1-GFP yeast strains determined by spot assay. For spot assay, cells were freshly streaked, grown overnight and then resuspended in normal saline to an A₆₀₀ of 0.1 (1×10⁶ cells) and 5 µl of five-fold serial dilutions, namely 1 (1∶5), 2 (1∶25), 3 (1∶125) and 4 (1∶625), of each strain was spotted on to YEPD plates in the absence (control) and presence of the following drugs: FLU (0.20 µg/ml), CYH (0.20 µg/ml), 4-NQO (0.20 µg/ml) and MTX (65 µg/ml). Growth differences were recorded following incubation of the plates for 48 hrs at 30°C. Growth was not affected by the presence of the solvents used for the drugs (data not shown). B: [³H] MTX and [³H] FLU accumulation in the different mutant variants of CaMdr1p-GFP. Controls AD1-8u⁻ and RPCaMDR1-GFP have also been included for comparison. The results are means±standard deviations for three independent experiments.

Figure 4. Protein expression profiles of CaMdr1p-GFP and its mutant variants in S. cerevisiae.

Panel A: Western Blot analysis of the PM fraction of mutant variants with anti-GFP antibody. B: Confocal and FACS analysis of the all the mutant variants to check their expression and localization in comparison with AD1-8u⁻ (negative control) and RPCaMDR1-GFP (positive control) .

Figure 5. The 3D homology model of CaMdr1p and the contact map derived from it.

Panel A: The 3D homology model of CaMdr1p wherein the mutated residues are marked onto the model and are coloured on the basis of the phenotypes exhibited upon mutation. Red denotes sensitive, green shows resistant and blue marks a position predicted to be important but not mutated as the CaMdr1p residues did not match the conserved residue in that alignment position. The structure is viewed using Visual Molecular Dynamic (VMD) software. B: The contact map of CaMdr1p is plotted between all the residues vs all the residues and displays the interactions between the beta carbon of each residue and beta carbon atoms of every other residue within and up to 8 A° of distance (Cα is used for glycine). Each cross points to an interaction between a residue on x-axis and a residue on y-axis. The lines represent the top thirty high RE_M residues and are coloured on the basis of the phenotypes where green represents the residues that are sensitive upon mutation while red are the ones that do not show any phenotype. The blue lines mark the residues in CaMdr1p that did not match with the most frequent residue in that particular column of the MSA.

Figure 6. Summary of inter-helical interactions via high RE_M residues.

Panel A: The table summarizes predicted inter-helical interactions mediated via selected residues with high RE_M. More than one residue pair is predicted to be involved in maintaining the interactions between the helices. B: Pictorial representation of inter-helical interactions via these high RE_M residues. Figure shows that the residues involved in these interactions are majorly confined to the N-terminal half of the protein.