title: Atomic modelling and systematic mutagenesis identify residues in multiple drug binding sites that are essential for drug resistance in the major Candida transporter Cdr1.
abstract: The ABC (ATP-Binding Cassette) transporter Cdr1 (Candida drug resistance 1) protein (Cdr1p) of Candida albicans, shows promiscuity towards the substrate it exports and plays a major role in antifungal resistance. It has two transmembrane domains (TMDs) comprising of six transmembrane helices (TMH) that envisage and confer the substrate specificity and two nucleotide binding domains (NBDs), interconnected by extracellular loops (ECLs) and intracellular loops (ICLs) Cdr1p. This study explores the diverse substrate specificity spectrum to get a deeper insight into the structural and functional features of Cdr1p. By screening with the variety of compounds towards an in-house TMH 252 mutant library of Cdr1p, we establish new substrates of Cdr1p. The localization of substrate-susceptible mutants in an ABCG5/G8 homology model highlights the common and specific binding pockets inside the membrane domain, where rhodamines and tetrazoliums mainly engage the N-moiety of Cdr1p, binding between TMH 2, 11 and surrounded by TMH 1, 5. Whereas, tin chlorides involve both N and C moieties located at the interface of TMH 2, 11, 1 and 5. Further, screening of the in house TMH mutant library of Cdr1p displays the TMH12 interaction with tetrazolium chloride, trimethyltin chloride and a Ca2+ionophore, A23187. In silico localization reveals a binding site at the TMH 12, 9 and 10 interface, which is widely exposed to the lipid interface. Together, for the first time, our study shows the molecular localization of Cdr1p substrates-binding sites and demonstrates the participation of TMH12 in a peripheral drug binding site.
authors: School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
authors: Shweta
authors: S
authors: Nim
authors: Drug Resistance & Membrane proteins lab, Molecular Microbiology and Structural Biochemistry CNRS-Lyon 1 University Research Unit n° 5086, Institut de Biologie et Chimie des Protéines, Lyon, France.
authors: Lucia Gonzalez
authors: LG
authors: Lobato
authors: Drug Resistance & Membrane proteins lab, Molecular Microbiology and Structural Biochemistry CNRS-Lyon 1 University Research Unit n° 5086, Institut de Biologie et Chimie des Protéines, Lyon, France.
authors: Alexis
authors: A
authors: Moreno
authors: Drug Resistance & Membrane proteins lab, Molecular Microbiology and Structural Biochemistry CNRS-Lyon 1 University Research Unit n° 5086, Institut de Biologie et Chimie des Protéines, Lyon, France.
authors: Vincent
authors: V
authors: Chaptal
authors: School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
authors: Manpreet Kaur
authors: MK
authors: Rawal
authors: Drug Resistance & Membrane proteins lab, Molecular Microbiology and Structural Biochemistry CNRS-Lyon 1 University Research Unit n° 5086, Institut de Biologie et Chimie des Protéines, Lyon, France. Electronic address: pierre.falson@ibcp.fr.
authors: Pierre
authors: P
authors: Falson
authors: Amity Institute of Integrative Sciences and Health, Amity University Haryana, Gurgaon, 122413, Haryana, India. Electronic address: rp47jnu@gmail.com.
authors: Rajendra
authors: R
authors: Prasad
mesh_terms: D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism; D000935:Antifungal Agents / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites; D000001:Calcimycin / Q000378:metabolism / Q000494:pharmacology; D002176:Candida albicans / Q000737:chemistry; D026141:Drug Resistance, Multiple, Fungal / Q000235:genetics*; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D062105:Molecular Docking Simulation; D016296:Mutagenesis; D009154:Mutation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012235:Rhodamines / Q000378:metabolism / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D013777:Tetrazoles / Q000378:metabolism / Q000494:pharmacology; D017971:Tin Compounds / Q000378:metabolism / Q000494:pharmacology
chemical_list: D000596:Amino Acids; D000935:Antifungal Agents; C094693:CDR1 protein, Candida albicans; D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; D012235:Rhodamines; D013777:Tetrazoles; D017971:Tin Compounds; C023599:stannous chloride; D000001:Calcimycin
keywords: ABC transporter; Candida albicans; Fungal drug resistance; Homology modelling; Protein drug interaction; Transmembrane helix 12