The barnacle Amphibalanus amphitrite is a major marine fouling organism that adheres strongly to submerged surfaces through proteinaceous cement. Among its adhesive proteins, CP19k plays a crucial role in stable underwater adhesion due to its amyloid-like β-sheet nanofibrils.Remarkably, AaCP19k has inspired biomimetic strategies to develop next-generation adhesives for biomedical applications despite immunogenicity challenges. Notably, a targeted computational strategy for the de-immunization of AaCP19k has not yet been reported; thus, this study addresses this significant research gap, presenting the first rational design of a de-immunized AaCP19k variant tailored for biocompatible underwater adhesion. Herein, we employed a computational workflow for protein engineering, combining epitope mapping, structural modeling, and molecular dynamics (MD) simulations to optimize stability and reduce immunogenicity of AaCP19k. Immunogenic regions were identified using IEDB MHC-II Binding Predictions and DiscoTope 2.0, followed by rational modifications through alanine scanning and β-hairpin redesign to minimize immunogenicity while preserving structural integrity. The engineered variants were modeled using AlphaFold2, then MD simulations in GROMACS were performed to evaluate the stability, flexibility, and conformational dynamics of the engineered variants compared to the native protein. Results revealed that targeted modifications to immunogenic regions significantly reduced predicted antigenicity. AlphaFold2 modeling confirmed preservation of the β-sandwich fold in the engineered protein with high confidence (average pLDDT = 94.6). MD simulations further validated these modifications, demonstrating enhanced structural stability with an average RMSD decrease of ~1.4 Å compared to the native protein. Additionally, RMSF analysis indicated reduced local flexibility, particularly in previously immunogenic regions within the β-hairpin between β6 and β7 (residues 143–166), as well as 110–112, 118–120. PCA and dynamic cross-correlation analysis supported overall structural stabilization. These findings demonstrate the effectiveness of our computational design strategy in optimizing AaCP19k as a promising bioadhesive candidate with reduced predicted immunogenicity, supporting its potential industrial and biomedical applications.
Zargari et al. (Sun,) studied this question.