Purpose: Calcineurin orchestrates growth, stress responses and virulence in major pathogenic fungi including Aspergillus fumigatus responsible for life-threatening fungal infections worldwide. While these cellular regulatory functions of calcineurin make it an attractive antifungal target, the immunosuppressive effects of the currently available calcineurin inhibitors, FK506 and CsA, make it difficult to exploit the antifungal potential due to conservation of calcineurin in the host and the fungal pathogen. Critical molecular understanding of calcineurin-immunophilin-immunosuppressor complexes would facilitate the design of novel non-immunosuppressive CsA and FK506 analogs for fungal-specific targeting of calcineurin.
Methods: We solved the crystal structure of calcineurin-FK506-FKBP12 complex in A. fumigatus and using site-directed mutagenic approaches, we constructed several mutations in the CnaA catalytic subunit of calcineurin and FKBP12. To identify differences between the A. fumigatus FKBP12 and Human FKBP12, we performed heterologous expression and mutations of Human FKBP12 in A. fumigatus. FKBP12 constructs were GFP tagged to visualize the binding of FKBP12 to calcineurin in the presence of FK506 in vivo by fluorescence microscopy. Molecular modeling and NMR analysis along with molecular dynamic simulations were performed to substantiate our structural and mutagenesis
Results: Human FKBP12 bound to A. fumigatus calcineurin in the presence of FK506 but did not inhibit the function of A. fumigatus calcineurin. However, mutation of specific residues in the 40s and 80s loop in human FKBP12 induced FK506 sensitivity. Furthermore, important A. fumigatus FKBP12 residues in the 40s and 80s loop required for the inhibition of calcineurin were identified. In addition, NMR studies on FK506-FKBP12-binding, and molecular dynamic simulations of the A. fumigatus calcineurin-FK506-FKBP12 complex based on the crystal structures, revealed that a key Phe residue at position 88 (F88) in 80s loop of A. fumigatus FKBP12 that is not conserved in human FKBP12 is essential for binding and inhibiting fungal calcineurin.
Conclusion: Our study for the first time provides the structural basis for the mechanism of inhibition of A. fumigatus calcineurin by FK506-FKBP12 complex. The identification of specific differences between human FKBP12 and A. fumigatus FKBP12 with respect to calcineurin inhibition broadens insight into developing novel non-immunosuppressive calcineurin inhibitors for effective antifungal targeting.
Full conference title:
- AAA 8th (2018)