Structural and functional analysis of CFEM proteins for hemoglobin iron acquisition

Candida albicans is a human commensal microorganism that can cause life-threatening systemic infections in immunocompromised individuals. The human host invests substantial efforts into withdrawing iron from potential pathogens. To overcome this, C. albicans has evolved a pathway for heme-iron scavenging from hemoglobin. This pathway includes a network of secreted and GPI-anchored extracellular proteins containing the cysteine-rich CFEM domain, which can extract heme from hemoglobin and transfer the heme across the cell envelope from one CFEM protein to the next, until delivery to the endocytic pathway. The crystal structure of the secreted CFEM protein Csa2 reveals that the CFEM domain adopts a novel helical-basket fold consisting of six α-helices, stabilized by four disulfide bond between eight conserved cysteine residues. Site-directed mutants of these cysteines suggest they play no role in heme binding or transfer. The planar heme molecule is bound between a flat hydrophobic platform located on top of the helical basket and a flexible N-terminal loop. Uniquely, an aspartic acid residue serves as the axial heme-iron ligand. Mutational analysis of the Csa2 protein surface identified mutants that are able to bind heme and extract it from hemoglobin, but that are defective in heme transfer. These mutants may identify a site of interaction between CFEM proteins that mediates heme transfer. Our results, which reveal the molecular details of the mechanism used by a fungal pathogen to overcome host nutritional immunity, may enable the development of novel antifungal therapies.