Redesign of the Ferritin Ferroxidase Center for Universal Molecular Binding or Specific Recognition

Although ferritin, as a versatile nanocarrier, has been engineered to improve cargo loading efficiency for various functions, including therapeutic applications, a universal design strategy enabling tunable molecular binding remains an unmet challenge. This study reports an AI-aided structure-guided engineering approach targeting the ferroxidase center of recombinant human heavy-chain ferritin (rHuHF), aiming to achieve either universal molecular binding or high-affinity specific recognition. Through site-directed mutagenesis of key residues within and flanking the ferroxidase center, two rHuHF variants (rHuHF-C1 and rHuHF-C2) were generated. X-ray crystallographic analysis revealed that the engineered pocket within rHuHF-C2 can accommodate a broad range of hydrophobic molecules (e.g., Curcumin, CUR) via hydrophobic interactions, thus validating their universal molecular binding capability. On the other hand, leveraging AI-assisted rational design, a variant (rHuHF-71) was subsequently engineered to specifically bind CUR with enhanced affinity, facilitated by the formation of hydrogen bonds and optimized hydrophobic contacts. This work establishes a generalizable strategy, designated as "Excavation, Rebuilding, and Validation", for engineering ferritin nanocages with tunable binding specificities, which holds great promise for advancing the development of protein-based drug delivery systems and the design of small-molecule binding proteins.