The immunosuppressant tacrolimus (FK506) inhibits C. glabrata Cdr1 efflux pump function by stabilizing the inward-facing conformation

Abstract The pathogenic yeast Candida glabrata is intrinsically resistant to azole antifungals through the overexpression of the multidrug transporter Cdr1. CgCdr1 detoxifies the yeast by expelling azoles out of the cell, thereby decreasing their intracellular concentration. Tacrolimus (FK506), one of the most widely used immunosuppressant medications used world-wide, has been identified as a broad-spectrum inhibitor of Cdr1 homologs in several Candida species. However, its mechanism of action remains unknown. We solved the cryoEM structure of CgCdr1 in complex with FK506, with or without ATP. The structure revealed that FK506 binds within the drug-binding site of CgCdr1, occupying the space occupied by Itraconazole. The hydrophobic face of FK506 stacks against the TMD1 and forms hydrogen bonds with TMD2, stabilizing a different conformation from the one adopted in FK-binding-proteins. FK506 binding triggered structural rearrangements bringing the nucleotide-binding-domains closer to the trans-membrane-domains, while stabilizing the inward-facing conformation. While ATP can still bind to the catalytic nucleotide-binding site, FK506 prevents the conformational transition required for ATP hydrolysis, thereby effectively blocking azole transport. Inter-particle variability analysis (3DVA) revealed significant conformational flexibility of FK506 within the binding pocket, with minimal transporter mobility. It allowed to visualize the conformational space occupied by the inhibitor within its binding-pocket, serving as a useful tool for inhibitor rational design. Overall, these findings demonstrate that FK506’s inhibition extends beyond competitive binding, involving allosteric modulation of the ATPase cycle. Significance statement The pathogenic yeast Candida glabrata exhibits intrinsic resistance to azole antifungals via the multidrug transporter Cdr1, which expels azoles from the cell. Tacrolimus (FK506), a widely used immunosuppressant, inhibits Cdr1 homologs across Candida species, yet its mechanism remained unknown. Here, we resolved the cryoEM structure of Cg Cdr1 in complex with FK506, revealing that FK506 binds to the drug-binding site, like itraconazole. Its hydrophobic face interacts with TMD1, while hydrogen bonds form with TMD2. FK506 stabilizes the inward-facing conformation, preventing ATP hydrolysis despite ATP binding, thereby blocking azole transport. Variability analysis highlighted FK506’s conformational flexibility within the pocket, offering insights for rational inhibitor design. These findings demonstrate that FK506’s inhibition involves both competitive binding and allosteric modulation of the ATPase cycle.