Rational Mutational Analysis of a Multidrug Mfs Transporter Camdr1P of Candida Albicans by Employing a Membrane Environment Based Computational Approach.

Rational Mutational Analysis of a Multidrug MFS Transporter CaMdr1p of Candida albicans by Employing a Membrane Environment Based Computational Approach
Khyati Kapoor1., Mohd Rehan2., Ajeeta Kaushiki2, Ritu Pasrija1¤, Andrew M. Lynn2*, Rajendra Prasad1*
1 School of Life Sciences, Jawaharlal Nehru University, New Delhi, India, 2 School of Information Technology, 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 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

References: 1. Prasad R, Kapoor K (2005) Multidrug resistance in yeast Candida. Int Rev Cytol 242: 215–248. 2. Gaur M, Choudhury D, Prasad R (2005) Complete inventory of ABC proteins in human pathogenic yeast, Candida albicans. J Mol Microbiol Biotechnol 9: 3–15. 3. Gaur M, Puri N, Manoharlal R, Rai V, Mukhopadhayay G, et al. (2008) MFS transportome of the human pathogenic yeast Candida albicans. BMC Genomics 9: 579. 4. Smriti, Krishnamurthy S, Dixit BL, Gupta CM, Milewski S, et al. (2002) ABC transporters Cdr1p, Cdr2p and Cdr3p of a human pathogen Candida albicans are general phospholipid translocators. Yeast 19: 303–318. 5. De RE, Arrigo P, Bellinzoni M, Silva PA, Martin C, et al. (2002) The multidrug transporters belonging to major facilitator superfamily in Mycobacterium tuberculosis. Mol Med 8: 714–724. 6. Ginn SL, Brown MH, Skurray RA (2000) The TetA(K) tetracycline/H(+) antiporter from Staphylococcus aureus: mutagenesis and functional analysis of motif C. J Bacteriol 182: 1492–1498. 7. Paulsen IT, Brown MH, Skurray RA (1996) Proton-dependent multidrug efflux systems. Microbiol Rev 60: 575–608. 8. Law CJ, Maloney PC, Wang DN (2008) Ins and outs of major facilitator superfamily antiporters. Annu Rev Microbiol 62: 289–305. PLoS Computational Biology | www.ploscompbiol.org 10 December 2009 | Volume 5 | Issue 12 | e1000624 Mutational Analysis of a Multidrug MFS Transporter 9. Abramson J, Smirnova I, Kasho V, Verner G, Kaback HR, et al. (2003) Structure and mechanism of the lactose permease of Escherichia coli. Science 301: 610–615. 10. Huang Y, Lemieux MJ, Song J, Auer M, Wang DN (2003) Structure and mechanism of the glycerol-3-phosphate transporter from Escherichia coli. Science 301: 616–620. 11. Yin Y, He X, Szewczyk P, Nguyen T, Chang G (2006) Structure of the multidrug transporter EmrD from Escherichia coli. Science 312: 741–744. 12. Yang Q, Wang X, Ye L, Mentrikoski M, Mohammadi E, et al. (2005) Experimental tests of a homology model for OxlT, the oxalate transporter of Oxalobacter formigenes. Proc Natl Acad Sci U S A 102: 8513–8518. 13. Sa-Correia I, dos Santos SC, Teixeira MC, Cabrito TR, Mira NP (2009) Drug:H+ antiporters in chemical stress response in yeast. Trends Microbiol 17: 22–31. 14. Pao SS, Paulsen IT, Saier MH Jr (1998) Major Facilitator Superfamily. Microbiol Mol Biol Rev 62: 1–34. 15. Egner R, Bauer BE, Kuchler K (2000) The transmembrane domain 10 of the yeast Pdr5p ABC antifungal efflux pump determines both substrate specificity and inhibitor susceptibility. Mol Microbiol 35: 1255–1263. 16. Shukla S, Robey RW, Bates SE, Ambudkar SV (2006) The calcium channel blockers, 1,4-dihydropyridines, are substrates of the multidrug resistance-linked ABC drug transporter, ABCG2. Biochemistry 45: 8940–8951. 17. Tutulan-Cunita AC, Mikoshi M, Mizunuma M, Hirata D, Miyakawa T (2005) Mutational analysis of the yeast multidrug resistance ABC transporter Pdr5p with altered drug specificity. Genes Cells 10: 409–420. 18. Ernst R, Kueppers P, Klein CM, Schwarzmueller T, Kuchler K, et al. (2008) A mutation of the H-loop selectively affects rhodamine transport by the yeast multidrug ABC transporter Pdr5. Proc Natl Acad Sci U S A 105: 5069–5074. 19. Saini P, Prasad T, Gaur NA, Shukla S, Jha S, et al. (2005) Alanine scanning of transmembrane helix 11 of Cdr1p ABC antifungal efflux pump of Candida albicans: identification of amino acid residues critical for drug efflux. J Antimicrob Chemother 56: 77–86. 20. Puri N, Gaur M, Sharma M, Shukla S, Ambudkar SV, et al. (2009) The amino acid residues of transmembrane helix 5 of multidrug resistance protein CaCdr1p of Candida albicans are involved in substrate specificity and drug transport. Biochim Biophys Acta 1788: 1752–1761. 21. Krogh A, Larsson B, von HG, Sonnhammer EL (2001) Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305: 567–580. 22. Ng PC, Henikoff JG, Henikoff S (2000) PHAT: a transmembrane-specific substitution matrix. Bioinformatics 16: 760–766. 23. Valdar WS (2002) Scoring residue conservation. Proteins 48: 227–241. 24. Cover T, Thomas J (2009) Elements of Information Theory. 25. Shannon CE (1997) The mathematical theory of communication. 1963. MD Comput 14: 306–317. 26. Wang K, Samudrala R (2006) Incorporating background frequency improves entropy-based residue conservation measures. BMC Bioinformatics 7: 385. 27. Hannenhalli SS, Russell RB (2000) Analysis and prediction of functional subtypes from protein sequence alignments. J Mol Biol 303: 61–76. 28. Srivastava PK, Desai DK, Nandi S, Lynn AM (2007) HMM-ModE improved classification using profile hidden Markov models by optimising the discrimination threshold and modifying emission probabilities with negative training sequences. BMC Bioinformatics 8: 104. 29. Reva B, Antipin Y, Sander C (2007) Determinants of protein function revealed by combinatorial entropy optimization. Genome Biol 8: R232. 30. Li L, Shakhnovich EI, Mirny LA (2003) Amino acids determining enzymesubstrate specificity in prokaryotic and eukaryotic protein kinases. Proc Natl Acad Sci U S A 100: 4463–4468. 31. Fischer JD, Mayer CE, Soding J (2008) Prediction of protein functional residues from sequence by probability density estimation. Bioinformatics 24: 613–620. 32. Capra JA, Singh M (2008) Characterization and prediction of residues determining protein functional specificity. Bioinformatics 24: 1473–1480. 33. Decottignies A, Grant AM, Nichols JW, De Wet H, McIntosh DB, et al. (1998) ATPase and multidrug transport activities of the overexpressed yeast ABC protein Yor1p. J Biol Chem 273: 12612–12622. 34. Nakamura K, Niimi M, Niimi K, Holmes AR, Yates JE, et al. (2001) Functional expression of Candida albicans drug efflux pump Cdr1p in a Saccharomyces cerevisiae strain deficient in membrane transporters. Antimicrob Agents Chemother 45: 3366–3374. 35. Pasrija R, Banerjee D, Prasad R (2007) Structure and function analysis of CaMdr1p, a major facilitator superfamily antifungal efflux transporter protein of Candida albicans: identification of amino acid residues critical for drug/H+ transport. Eukaryot Cell 6: 443–453. 36. Pirovano W, Feenstra KA, Heringa J (2008) PRALINETM: a strategy for improved multiple alignment of transmembrane proteins. Bioinformatics 24: 492–497. 37. Ditty JL, Harwood CS (1999) Conserved cytoplasmic loops are important for both the transport and chemotaxis functions of PcaK, a protein from Pseudomonas putida with 12 membrane-spanning regions. J Bacteriol 181: 5068–5074. 38. Guan L, Kaback HR (2006) Lessons from lactose permease. Annu Rev Biophys Biomol Struct 35: 67–91. 39. Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32: 1792–1797. 40. Henikoff S, Henikoff JG (1992) Amino acid substitution matrices from protein blocks. Proc Natl Acad Sci U S A 89: 10915–10919. 41. Chandrasekaran A, Ojeda AM, Kolmakova NG, Parsons SM (2006) Mutational and bioinformatics analysis of proline- and glycine-rich motifs in vesicular acetylcholine transporter. J Neurochem 98: 1551–1559. 42. Martin LC, Gloor GB, Dunn SD, Wahl LM (2005) Using information theory to search for co-evolving residues in proteins. Bioinformatics 21: 4116–4124. 43. Koike K, Oleschuk CJ, Haimeur A, Olsen SL, Deeley RG, et al. (2002) Multiple membrane-associated tryptophan residues contribute to the transport activity and substrate specificity of the human multidrug resistance protein, MRP1. J Biol Chem 277: 49495–49503. 44. Schiffer M, Chang CH, Stevens FJ (1992) The functions of tryptophan residues in membrane proteins. Protein Eng 5: 213–214. 45. Chen CP, Kernytsky A, Rost B (2002) Transmembrane helix predictions revisited. Protein Sci 11: 2774–2791. 46. Shukla S, Saini P, Smriti, Jha S, Ambudkar SV, et al. (2003) Functional characterization of Candida albicans ABC transporter Cdr1p. Eukaryot Cell 2: 1361–1375. 47. Sali A, Blundell TL (1993) Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 234: 779–815. 48. Barton GJ (1993) ALSCRIPT: a tool to format multiple sequence alignments. Protein Eng 6: 37–40. 49. Livingstone CD, Barton GJ (1993) Protein sequence alignments: a strategy for the hierarchical analysis of residue conservation. Comput Appl Biosci 9: 745–756. 50. Sigal N, Molshanski-Mor S, Bibi E (2006) No single irreplaceable acidic residues in the Escherichia coli secondary multidrug transporter MdfA. J Bacteriol 188: 5635–5639. PLoS Computational Biology | www.ploscompbiol.org 11 December 2009 | Volume 5 | Issue 12 | e1000624