Cryo-EM structures of Candida albicans Cdr1 reveal azole-substrate recognition and inhibitor blocking mechanisms

Abstract

In Candida albicans, Cdr1 pumps azole drugs out of the cells to reduce intracellular accumulation at detrimental concentrations, leading to azole-drug resistance. Milbemycin oxime, a veterinary anti-parasitic drug, strongly and specifically inhibits Cdr1. However, how Cdr1 recognizes and exports azole drugs, and how milbemycin oxime inhibits Cdr1 remain unclear. Here, we report three cryo-EM structures of Cdr1 in distinct states: the apo state (Cdr1^Apo), fluconazole-bound state (Cdr1^Flu), and milbemycin oxime-inhibited state (Cdr1^Mil). Both the fluconazole substrate and the milbemycin oxime inhibitor are primarily recognized within the central cavity of Cdr1 through hydrophobic interactions. The fluconazole is suggested to be exported from the binding site into the environment through a lateral pathway driven by TM2, TM5, TM8 and TM11. Our findings uncover the inhibitory mechanism of milbemycin oxime, which inhibits Cdr1 through competition, hindering export, and obstructing substrate entry. These discoveries advance our understanding of Cdr1-mediated azole resistance in C. albicans and provide the foundation for the development of innovative antifungal drugs targeting Cdr1 to combat azole-drug resistance.

Fig. 1. Functional and cryo-EM studies of C. albicans Cdr1.

a A schematic diagram of efflux pumps in C. albicans. Cdr1, Cdr2 and Mdr1 can expel azole antifungal drugs. Milbemycin inhibits Cdr1 and Cdr2 efflux activity. The green arrows indicate the direction of azole drug efflux. b Fluconazole-sensitivity assay by measuring OD_600nm under different fluconazole concentrations. The IC₅₀ value for the empty vector is 0.31 µg/mL (left figure), while Cdr1 overexpression raises the IC₅₀ value to 56.6 µg/mL (right figure). Data are presented as mean values ± SD; n = 3 independent experiments. c Milbemycin oxime-sensitivity assay measuring OD_600nm under different milbemycin oxime concentrations, with fluconazole concentrations of 0 µg/mL, 1 µg/mL, and 10 µg/mL. The IC₅₀ values for 1 µg/mL and 10 µg/mL fluconazole are 0.034 µg/mL and 0.009 µg/mL, respectively. Data are presented as mean values ± SD; n = 3 independent experiments. d Overall structures of Cdr1^Apo, Cdr1^Flu and Cdr1^Mil. TMD1 and NBD1 are depicted in sky-blue, while TMD2 and NBD2 are in orange. PIP2, fluconazole, and milbemycin oxime are colored by yellow, red, and green, respectively. The green shading represents plasma membrane region.

Fig. 2. Fluconazole recognition by TMDs of Cdr1.

a Sectional view displaying the electrostatic surface of Cdr1^Flu. Red-colored fluconazole resides at the top of the hydrophobic central cavity. b Side (left figure) and bottom (right figure) views of Cdr1^Flu. Fluconazole is surrounded by TM1b, TM2a and TM5a from TMD1(sky-blue), TM8 and TM11a from TMD2 (orange). c Coordination of fluconazole by TMDs of Cdr1^Flu. Residues from TMD1 are colored by sky-blue, while those from TMD2 are colored by orange. Magenta dashes indicate hydrogen bonds. d Relative OD_600nm values for wild type and mutants under a 10 µg/mL fluconazole concentration. The y-axis represents the relative OD_600nm values as a percentage of the control (no drug). Three independent experiments were conducted for both wild type and mutants. Data are presented as mean values ± SD; n = 3 independent experiments.

Fig. 3. Structural comparison of Cdr1^Flu and Pdr5^AOV.

a Side view (upper figure) and bottom view (lower figure) for structural alignment Cdr1^Flu and Pdr5^AOV (PDB: 7P06). Models of Cdr1^Flu and Pdr5^AOV are distinguished by using light-blue and wheat colors, respectively. b Zoomed-in view of TM1b and TM11a region of Cdr1^Flu and Pdr5^AOV. TM1b and TM11a move inward in Pdr5^AOV. c, Bottom view of TM1b, TM2b, TM8 and TM11 regions of Cdr1^Flu and Pdr5^AOV. The lower regions of these helices move inward in Pdr5^AOV. d Residues in TM2, TM8 and TM11 of Pdr5^AOV exhibit clashes with fluconazole. Phe562 in Pdr5^AOV prevents upward movement of fluconazole along the axis between TM2 and TM11. e Section view within the fluconazole binding region. TM2, TM5, TM8, and TM11 in Pdr5^AOV undergo an inward movement within the fluconazole binding site. f Top view of TM2a, TM5a, TM8 and TM11 regions of Cdr1^Flu and Pdr5^AOV. The upper regions of TM2a, TM5a, and TM8 move outward, while that of TM11 shifts inward. g The light-blue grid mesh depicts the exit channel of fluconazole in Pdr5^AOV. The red arrows indicate the movement direction of the helices.

Fig. 4. Milbemycin oxime recognition by TMDs of Cdr1.

a Sectional view displaying the electrostatic surface of Cdr1^Mil. Green-colored milbemycin oxime resides at the top of the hydrophobic central cavity. b Side (left figure) and bottom (right figure) views of Cdr1^Mil. Milbemycin oxime is surrounded by TM1b, TM2a and TM5a from TMD1(sky-blue), TM8 and TM11a from TMD2 (orange). c Coordination of milbemycin oxime by TMDs of Cdr1^Mil. Residues from TMD1 are colored by sky-blue, while those from TMD2 are colored by orange. Magenta dashes indicate hydrogen bonds. d Comparison of the TM1b region in Cdr1^Mil and Cdr1^Apo, with the lower portion of TM1b moving toward the central cavity due to hydrophobic interactions. The red arrows indicate the movement direction of TM1a and TM1b.

Fig. 5. Inhibitory mechanism of milbemycin oxime.

a Overlapped binding poses of milbemycin oxime and fluconazole. Steric clash explains their competition for binding to Cdr1. Fluconazole, and milbemycin oxime are colored by red and green, respectively. b Overlapped binding poses of milbemycin oxime in Cdr1^Mil and R6G in Pdr5^R6G (PDB: 7P05). Steric clash explains their competition for binding to Cdr1. Milbemycin oxime and R6G are colored by green and yellow, respectively. c Relative ATPase activity of wild type Cdr1 by adding DMSO, fluconazole (Flu), milbemycin oxime (Mil) or fluconazole with milbemycin oxime (Flu+Mil). The concentrations of fluconazole and/or milbemycin oxime were ten times greater than that of the purified Cdr1. These drugs are dissolved in DMSO. Data are presented as mean values ± SD; n = 3 independent experiments. Stability analysis of the cytoplasmic and inner-leaflet entrances of Cdr1^Flu (d) and Cdr1^Mil (e). The left panels in both figures are shown by B-factor analysis. Higher B-factors are depicted in red, indicating greater flexibility, while lower B-factors are shown in blue, signifying rigidity. The electrostatic potential is color-coded in the right panels, ranging from red (indicating negative charge) to blue (indicating positive charge). The dashed circle marks the cytoplasmic (black) and inner-leaflet (green) entrances in the panels. The orange arrows indicate the substrate entrance channel.

Fig. 6. Model for fluconazole efflux and inhibition by milbemycin oxime.

In the resting state, Cdr1 maintains an inward-open conformation, allowing the entry of hydrophilic fluconazole from the cytoplasm (dash purple arrow). As fluconazole enters the hydrophobic cavity, ATP molecules act as molecular glue to induce dimerization of NBDs and trigger a conformational change from inward to outward, leading to the release of fluconazole (dash green arrow). Following the hydrolysis of the ATP in NBD2, Cdr1 returns to its inward-facing conformation, starting the next cycle of fluconazole transport. Milbemycin oxime, upon entering the hydrophobic cavity, induces a conformational change that closes the entrances (purple arrow). It competes for binding at the central hydrophobic cavity, obstructing fluconazole. Additionally, the closure of the entrances effectively blocks the entry of fluconazole into this region (blocked dash purple arrow). Furthermore, milbemycin oxime acts as a molecular glue, locking the two TMDs, inhibiting ATPase activity, and preventing the transportation process.