Biochemical and structural analysis of Candida albicans multidrug efflux pump Cdr1

Pleiotropic drug resistance (PDR) ATP-binding cassette (ABC) transporters are abundant eukaryotic membrane proteins that pump a vast array of different compounds across organelle and cell membranes. Overexpression of the archetype fungal PDR transporter Cdr1 is the main cause of azole drug resistance in Candida albicans, a major fungal pathogen that can cause serious, life threatening, invasive fungal infections in immunocompromised individuals. Fluconazole, an azole antifungal, is commonly used to treat these infections. Azole resistance of C. albicans isolates, however, is of serious clinical concern with often fatal outcomes. An attractive approach to overcome azole resistance is the use of efflux pump inhibitors in combination with azoles. In order to achieve this result, understanding how these efflux pumps work and obtaining high-resolution structures will help to develop suitable inhibitors that do not easily give rise to resistance. Yet, to date, no structure for any PDR ABC transporter has been solved. PDR transporters are one of the largest membrane protein superfamilies. Many plants and fungi have more than ten, some up to 50, different PDR transporters; which indicates that they are likely to be important for their survival in complex, frequently changing, environments. As such, they are of great importance to human fungal disease, and in agriculture. The objectives of this project were to: i) investigate the role of cysteine amino acids in the stability, trafficking and function of C. albicans Cdr1; ii) hyperexpress Cdr1 in the eukaryotic model organism, Saccharomyces cerevisiae, and optimise the isolation and purification of Cdr1; and iii) attempt to determine the structure of Cdr1 with X-ray crystallography or cryo-EM. In this PhD project, a successful workflow was developed that led to the purification of 0.5 mg Cdr1 per litre of the culture medium in pure, stable, monodisperse form that was used to perform structural investigations of Cdr1 with X-ray crystallography and cryo-EM. In addition, 21 cysteine-deficient Cdr1 mutants were created, and biochemically characterised, which revealed that the six conserved extracellular cysteines were most critical for proper Cdr1 expression, localisation, and function. One fully functional ‘almost Cys-less’ version of Cdr1, Cdr1P-CID, with all but the six conserved extracellular cysteines replaced with serine, alanine or isoleucine was constructed. Cdr1P-CID will be critical in cysteine-crosslinking studies that will help confirm the biological significance of any future structure of Cdr1. An additional four mutants were constructed to generate catalytically inactive transporters. Two conserved nucleotide binding domain residues (D327 and E1027) contributing catalytic bases for ATP-hydrolysis, were replaced with asparagine or glutamine. In addition to those three mutants (Cdr1-D327N, -E1027Q, and -D327N-E1027Q), a fourth catalytically inactive Cdr1 mutant (Cdr1-K901A) was also generated. Detergent screening, using 31 detergents with different chemistries, revealed that n-dodecyl-β-D-maltopyranoside (DDM) and lauryl maltose neopentyl glycol (LMNG) worked best for Cdr1 solubilisation and/or purification. They provided purified monomeric Cdr1 molecules for crystallography and electron microscopy. Crystal trials using four different commercial crystal screens identified some conditions that resulted in small crystal formations that need to be optimised in order to grow bigger crystals. Protocols for negative stain EM and cryo-EM were optimised which resulted in the first very low-resolution (~18 Å) structures of negative stained detergent-purified Cdr1, however more work is needed to resolve ambiguities between the structures. This study has revealed important insights into the structure-function relationship of C. albicans Cdr1 and laid the foundations for obtaining high-resolution Cdr1 structures