Amino Acid Residue-Driven Nanoparticle Targeting of Protein Cavities: Surface Chemistry Dictates Binding Specificity beyond Size Complementarity

Cavities at protein-protein interaction interfaces are considered “undruggable” because their shallow or large geometries hinder stable binding by small molecules. Overcoming this limitation is essential for developing new therapies. Cerium oxide nanoparticles (CeO 2 NPs) of a suitable size can occupy the 5 nm central cavity of the SARS-CoV-2 spike (S) trimer, thereby inhibiting infection. Although size compatibility enables cavity access, other targeting parameters remain unclear. The S trimer features central and lateral cavities, both ∼5 nm in diameter, that can accommodate NPs. To explore factors governing cavity targeting, we compared size-matched CeO 2 NPs and gold NPs (AuNPs), evaluating their binding to S-trimer cavities and investigating mechanisms underlying their selective interactions. Despite comparable antiviral activity, the two NPs exhibited different binding profiles. Biolayer interferometry confirmed strong binding of both NPs to the S trimer, while only CeO 2 NPs selectively targeted the receptor-binding domain (RBD). CeO 2 NPs preferentially occupied the central cavity enriched with Asp residues. However, AuNPs bind to the lateral cavities, including the S1/S2 cleavage site, where they interact with Arg-rich motifs for furin-mediated activation of the S protein. Mechanistically, CeO 2 NPs achieved stable binding by coordinating with Asp carboxyl groups in the central cavity, while AuNPs bound lateral cavities primarily via electrostatic attraction with Arg residues. These findings suggest that residue-level recognition governs NP binding among sterically accessible protein cavities, with size compatibility enabling access and surface-residue matching defining binding selectivity. The insight provides a strategy for designing NPs to engage recessed features on protein surfaces previously inaccessible, enabling selective functional modulation and broader biomedical applications.