Eligible Student Poster 49th Lorne Conference on Protein Structure and Function 2024

Unveiling Candida albicans' Multi-Drug Resistance: A Biophysical and Structural Analysis of its ABC Transporter (#106)

Jeeeun Shin 1 , Jobichen Chacko 2 , Chung-Han Tsai 2 , Melanie Rug 2 , Joseph Brock 1
  1. Research School of Biology, The Australian National University , Canberra , ACT, Australia
  2. Centre for Advanced Microscopy (CAM), The Australian National University, Canberra, ACT, Australia

Despite the efficacy of topical remedies for less severe instances, unmanaged invasive candidiasis can lead to severe complications1. Moreover, it is crucial not to underestimate the significance of COVID-19-related candidiasis pathogenesis2. However, the increasing drug resistance, facilitated by the overexpression of Candida Drug Resistance Protein 1 (CDR1)3 among pathogenic Candida species presents a significant hurdle in candidiasis treatment. CDR1's ability to efflux diverse hydrophobic compounds, including lipids4, sterols5, and rhodamine dye molecules6, highlights its role as a versatile multi-drug resistance ABC transporter. Understanding the molecular mechanisms of substrate binding and efflux is crucial, especially in the context of resistance to commonly administered azole-type drugs7,8 We aimed to unravel the molecular intricacies of drug resistance by investigating the substrate-binding properties of CDR1 in Candida albicans (CalbCDR1), a prominent candidiasis contributor. We successfully expressed and purified mTurquoise2 fusion CalbCDR1 using Saccharomyces cerevisiae, where the expression and detergent conditions were rapidly screened by measuring the fluorescence9. Later, the mTurquoise2 tag was cleaved using TEV protease. Here, we present the Cryo-EM structure of fluconazole-bound CalbCDR1 in an inward-open conformation at a resolution of 3.4 Å. We found that the inward-open conformation allows access to various substrates into the hydrophobic binding pocket of its transmembrane domain (TMD), which ultimately leads to the efflux of the drug. Our findings additionally indicate that the conformational stability remains unaffected by the binding of fluconazole, as evidenced by a moderate rise in the melting temperature. Further biophysical investigation revealed that another known substrate, Rhodamine 6G, shows very weak binding. Substrates displaying low binding affinity to CalbCDR1 contribute to its multi-drug resistance characteristics, as such substrates can readily traverse the transmembrane domain without engaging in strong interactions. Nonetheless, the binding of dye molecules such as rhodamine allows us to take advantage of their fluorescent characteristics to develop a high-throughput drug screening assay. Our study provides a comprehensive understanding of CalbCDR1, elucidating its structural dynamics and substrate-binding properties. These findings offer vital insights for developing innovative antifungal strategies against drug-resistant Candida infections, addressing a critical healthcare concern. 

 

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