AtaA as a Self-Immobilizing Scaffold for Enzymatically Active Surfaces through Oxidative Tyrosine Coupling

Protein nanofibers are hold great promise as genetically programmable scaffolds for constructing functional material interfaces. AtaA, a giant trimeric autotransporter adhesin from Acinetobacter sp. Tol 5, forms an elongated architecture from the bacterial cell surface, enabling the cells to adhere directly to a wide variety of abiotic surfaces. However, the potential use of AtaA as a scaffold with additional biological functions remains largely unexplored. In this study, we investigated the use of AtaA as a self-immobilizing scaffold for constructing enzymatically active surfaces via horseradish peroxidase (HRP)-catalyzed tyrosine coupling. We prepared a recombinant AtaA nanofiber fragment (NhNs-AtaA) and its derivative containing two tyrosine-containing loop structures (YL-NhNs-AtaA). Both recombinant proteins retained their adhesive properties. Treatment with HRP and H2O2 generated high-molecular-weight products from both proteins, and dityrosine fluorescence emission indicated the formation of tyrosine-derived covalent cross-links. Y-Loop insertion further increased the consumption of monomeric AtaA and promoted the formation of high-molecular-weight products. The removal of arginine, which was included during purification, further increased the loss of monomeric AtaA in both constructs, indicating that intermolecular protein association influenced productive AtaA cross-linking. NhNs-AtaA and YL-NhNs-AtaA were combined with Y-tagged bacterial alkaline phosphatase (BAP-Y). HRP-mediated coupling generated high-molecular-weight products consistent with the cross-linked AtaA-BAP-Y conjugates and enabled the retention of BAP activity on AtaA-coated surfaces without prior chemical modification. Although Y-Loop insertion enhanced polymerization in solution, it reduced AtaA adhesion and consequently lowered surface-bound phosphatase activity, revealing a trade-off between cross-linking reactivity and native adhesive function. These findings establish AtaA as a self-immobilizing protein nanofiber scaffold for constructing enzymatically active surfaces, and suggest the potential extension of this strategy to other AtaA-based interfaces.