BIOPHYSICAL AND STRUCTURAL CHARACTERIZATION OF THE CrpA PROTEIN FROM Aspergillus fumigatus

Aspergillus fumigatus is a saprophytic fungus capable of surviving a broad range of adverse conditions, owing to mechanisms such as the production of highly stable hydrophobic conidia, a cell wall rich in chitin and melanin, and the synthesis of secondary metabolites. During human infection, it has evolved additional adaptations to cope with host-imposed stresses, including variations in temperature, pH and water balance, oxidative damage, and the action of antifungal molecules. The high mortality associated with infection underscores the need to elucidate the molecular mechanisms that enable the fungus to evade the immune response and establish disseminated and chronic infections. In this context, the cysteine-rich repeat proteins (Crp) constitute a family exclusive to A. fumigatus, involved in immune evasion, whose molecular function remains unknown. CrpA, recently identified among the conidial surface proteins, stands out as a relevant target, particularly owing to the novel fold of its N-terminal domain (CrpA_N). This singularity is reinforced by analysis in TED (The Encyclopedia of Domains), which extends the CATH database to include AlphaFold-generated structures lacking experimental data. In collaboration with Prof. Christine Orengo, CrpA_N was identified within a cluster of 44 sequences, mostly from Aspergillus species, sharing the same predicted fold, none of which has been experimentally resolved or functionally characterized. The aim of this work was to characterize CrpA structurally and biophysically. The full-length recombinant protein, comprising the N-terminal (CrpA_N) and C-terminal (CrpA_C) domains, was expressed in Escherichia coli Origami-2 (DE3) and purified by affinity and size-exclusion chromatography. The CrpA_N domain structure was determined by X-ray crystallography at 2.3 Å. The isolated domains were characterized by SEC-MALS, circular dichroism, DLS, and DSF, whereas the full-length protein was analyzed by mass photometry and cryo-electron microscopy (cryo-EM). The analyses revealed that CrpA predominantly forms a high-molecular-weight oligomeric complex; cryo-EM reconstruction indicated oligomers of up to dodecamers, consistent with the predominant population of approximately 540 kDa observed by mass photometry. Taken together, these results provide new insights into the structural organization of CrpA and establish a basis for understanding its biological function and its potential as an antifungal target.