Enzyme-Mimicking Active Site Clefts Demonstrated by Self-Assembled Peptide Nanoribbons with Polar Zippers

Due to the inherent limitations of natural enzymes, biomimetic enzymes have received tremendous attention, among which those arising from peptide self-assembly are of particular interest due to their resemblance to natural enzymes in composition and hierarchical structures, as well as their structural robustness and designability. Despite considerable advances achieved in this area, it remains a major challenge to construct active site clefts through peptide self-assembly. Here, we report the design of polar zippers between peptide β-sheets to mimic the catalytic microenvironment of natural enzymes. As a supersecondary structural motif stabilized by the side chain-side chain hydrogen bonding, polar zippers not only promote significant β-sheet lamination to form wide nanoribbons but also constitute clefts on the nanoribbons' surface. Among the three designed peptide analogues (I₃GH, I₃GHK, and I₃HGK), histidine (His or H) polar zippers between β-sheets form only in the self-assembly of I₃HGK, thus leading to the formation of wide I₃HGK nanoribbons and thin I₃GH and I₃GHK nanofibrils. Compared to the I₃GHK and I₃GH nanofibrils, the I₃HGK nanoribbons exhibit substantially increased catalytic efficiency in the hydrolysis of <i>p</i>-nitrophenyl acetate (<i>p</i>NPA) due to the synergistic interplay of polar reactive His residues and hydrophobic Ile(I) residues buried within the clefts. By substituting other uncharged polar residues for His within the clefts, the catalytic ability of the peptide nanoribbons can be tuned, with the I₃CGK ones exhibiting the highest catalytic efficiency in the <i>p</i>NPA hydrolysis, owing to the potent nucleophilicity of the cysteine (Cys or C) side chain. This work offers a new conceptual framework for mimicking the catalytic cleft of natural enzymes through the rational design and self-assembly of short peptides.