Pleiotropic drug resistance (PDR) ATP-binding cassette (ABC) transporters of the ABCG family are eukaryotic membrane proteins that pump an array of compounds across organelle and cell membranes. Overexpression of the archetype fungal PDR transporter Cdr1 is a major cause of azole antifungal drug resistance in Candida albicans, a significant fungal pathogen that can cause life-threatening invasive infections in immunocompromised individuals. To date, no structure for any PDR transporter has been solved. The objective of this project was to investigate the role of the 23 Cdr1 cysteine residues in the stability, trafficking, and function of the protein when expressed in the eukaryotic model organism, Saccharomyces cerevisiae. The biochemical characterization of 18 partially cysteine-deficient Cdr1 variants revealed that the six conserved extracellular cysteines were critical for proper expression, localization, and function of Cdr1. They are predicted to form three covalent disulfide bonds that stabilize the large extracellular domains of fungal PDR transporters. Our investigations also revealed a novel nucleotide-binding domain motif, GX_2[3]CPX₃NPAD/E, at the peripheral cytosolic apex of ABCG transporters that possibly contributes to the unique ABCG transport cycle. With this knowledge, we engineered an “almost cysteine-less,” yet fully functional, Cdr1 variant, Cdr1P-CID, that had all but the six extracellular cysteines replaced with serine, alanine, or isoleucine (C1106I of the new motif). It is now possible to perform cysteine-cross-linking studies that will enable more detailed biochemical investigations of fungal PDR transporters and confirm any future structure(s) solved for this important protein family.

中文翻译：

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设计半胱氨酸缺乏的功能性白色念珠菌 Cdr1 分子揭示了 ABCG 转运蛋白胞质顶点的保守区域，这对 Cdr1 的正确折叠和运输很重要

ABCG 家族的多效耐药性 (PDR) ATP 结合盒 (ABC) 转运蛋白是真核膜蛋白，可将一系列化合物泵送穿过细胞器和细胞膜。原型真菌 PDR 转运蛋白 Cdr1 的过度表达是白色念珠菌中唑类抗真菌药物耐药性的主要原因，白色念珠菌是一种重要的真菌病原体，可在免疫功能低下的个体中引起危及生命的侵袭性感染。迄今为止，尚未解决任何 PDR 转运蛋白的结构。该项目的目的是研究 23 个 Cdr1 半胱氨酸残基在真核模型生物酿酒酵母中表达时在蛋白质的稳定性、运输和功能中的作用. 18 个部分缺乏半胱氨酸的 Cdr1 变体的生化特征表明，六个保守的细胞外半胱氨酸对于 Cdr1 的正确表达、定位和功能至关重要。预计它们会形成三个共价二硫键，稳定真菌 PDR 转运蛋白的大细胞外结构域。我们的研究还揭示了一个新的核苷酸结合域基序，GX _2[3] CPX ₃NPAD/E，位于 ABCG 转运蛋白的外周胞质顶点，可能有助于独特的 ABCG 转运循环。有了这些知识，我们设计了一个“几乎不含半胱氨酸”但功能齐全的 Cdr1 变体，Cdr1P-CID，除了六个细胞外半胱氨酸外，其他所有半胱氨酸都被丝氨酸、丙氨酸或异亮氨酸（新基序的 C1106I）取代。现在可以进行半胱氨酸交联研究，这将能够对真菌 PDR 转运蛋白进行更详细的生化研究，并确认为这个重要的蛋白质家族解决的任何未来结构。