Novel motif in calcineurin catalytic subunit is required for septal localization of calcineurin in Aspergillus fumigatus

Abstract

Calcineurin heterodimer, comprised of the catalytic (CnaA) and regulatory (CnaB) subunits, localizes at the hyphal tips and septa to direct growth, septation, and disease in the human pathogen Aspergillus fumigatus. Here we discovered a novel motif (FMDVF) required for this critical CnaA septal localization, including residues Phe368, Asp370 and Phe372 overlapping the cyclosporine A-cyclophilin A-binding domain, CnaB-binding helix and the FK506-FKBP12-binding pocket. Mutations in adjacent residues Asn367, Trp374, and Ser375 confer FK506 resistance without impacting CnaA septal localization. Modeling A. fumigatus CnaA confirmed that the FMDVF motif forms a bridge between the two known substrate-binding motifs, PxIxIT and LxVP, and concurrent mutations (F368A D370A; F368A F372A) in the FMDVF motif disrupt CnaA-substrate interaction at the septum.

Keywords: Aspergillus fumigatus; FK506; LxVP motif; PxIxIT motif; calcineurin; septum.

Figure 1

(A) Schematic of domains of A. fumigatus calcineurin A: residues in the cyclosporine A-cyclophilin A binding domain (red underline), the FK506-FKBP12 binding domain (blue underline) and the CnaB-binding helix (CnBBH; green underline). PxIxIT-binding NIR residues are in bold and FMDVF motif residues underlined. Catalytic domain is orange, and the remaining C-terminal portion, including the CnBBH, the calmodulin-binding domain (CaMBD) and the autoinhibitory domain (AID), are boxed. (B) Radial growth of the wild-type strain (akuB^KU80) and the CnaA FMDVF mutant strains (CnaA-F368A; CnaA-D370A; CnaA-F368A D370A and CnaA-F368A F372A) assessed after 5 days and depicted as mean ± standard deviation. (C) Respective FMDVF mutant strains cultured on coverslips in GMM liquid medium for 18–20 h at 37 °C. Scale bar is 10 μm.

Figure 2

(A) Strains expressing FMDVF mutated CnaA-EGFP constructs cultured in GMM liquid medium on coverslips for 18–20 h. Localization is indicated as septal, partial septal or cytosolic. Arrowheads indicate proper localization of CnaA-EGFP on either side of the hyphal septa. Dotted arrow indicates aberrantly localized CnaA-EGFP at the hyphal septa following CnaA-F368A D370A mutation. White arrow indicates complete mislocalization of CnaA-EGFP from the hyphal septum upon CnaA-F368A F372A mutation. A total of 100 septa were observed in each case. Scale bar is 10 μm. (B) Western detection performed using the anti-GFP polyclonal primary antibody and peroxidase labeled anti-rabbit IgG secondary antibody. Arrow indicates the ~92 kDa CnaA-EGFP fusion protein. (C) Clustal alignment of the region encompassing the FMDVF motif showing conservation between higher and lower eukaryotes. The FMDVF motif is boxed in yellow. Hs-Homo sapiens; Sc-Saccharomyces cerevisiae; Sp-Schizosaccharomyces pombe; Cn-Cryptococcus neoformans; Af-Aspergillus fumigatus; Nc-Neurospora crassa.

Figure 3

(A) Mutations conferring FK506 resistance. Control strain (akuB^KU80), CnaA-N367D, CnaA-W374L and CnaA-S375T strains cultured in the absence (Control; upper panel) or presence of calcineurin inhibitors FK506 (100 ng/ml; middle panel) and cyclosporine A (10 μg/ml) for 5 days. (B) 1×10⁴ conidia of each strain inoculated into 200 μl of RPMI liquid medium in the absence or presence of FK506 (100 ng/ml), photographs (×10 magnification) taken after 24 h of growth. (C) FMDVF mutant strains examined for FK506 (100 ng/ml) susceptibility on GMM agar medium for 5 days. 1×10⁴ conidia of each strain inoculated. Experiments were repeated three times, each in triplicate.

Figure 4

(A) PxIxIT and LxVP substrate binding motifs with various mutations in the FMDVF motif indicated in black arrows and other mutations in the adjacent residues indicated by dotted arrows (B) Modeling functional motifs of calcineurin A from A. fumigatus and their importance for protein-substrate communication. The X-ray structure (PDB 4F0Z) of human calcineurin A (purple, CnaA) and B (pink, CnaB) bound to a viral peptide regulator (A238L; green). Both LxVP and PxIxIT binding motifs indicated and surfaces colored dark green. (C, D) Detailed interactions of the LxVP motif and calcineurin are shown. Residues with an asterisk numbered according to the A. fumigatus CnaA sequence. Molecular surfaces are of only those residues that contribute to regulator interactions and colored according to CnaA domain affiliation. The LxV portion of the regulator peptide motif interdigitates with F378* and W374* residues located on the CnaA α-tower and packs against Y363* (upper). Same binding position for FK506 is also shown from the X-ray structure (PDB 1TCO) of bovine calcineurin in complex with FK506 (lower). (D) The PxIxIT motif interacts with the NIR signature sequence of CnaA and forms an extended β-sheet. (E) Cartoon illustrates relationship between the functional motifs of substrate and CnaA domains. Bridging the LxVP and PxIxIT site is the FMDVF motif that forms the foundation for orienting the α-tower. Shown in inset figure, F368* packs into a shallow hydrophobic pocket on the surface of CnaA, whereas F372* interacts with a hydrophobic groove at the base of CnaB. D370* directly coordinates F372* and T373* orienting the N-terminal end of the α-tower.