Structure–activity analysis of buforin II, a histone H2A-derived antimicrobial peptide: The proline hinge is responsible for the cell-penetrating ability of buforin II

TLDR

Buforin II is a 21‑residue antimicrobial peptide that adopts an amphipathic structure comprising an N‑terminal random coil, an extended helix, a hinge, and a C‑terminal α‑helix. The study aimed to identify structural elements of buforin II essential for its antimicrobial activity by synthesizing truncated or substituted analogs and assessing their potency and mechanism of action. The authors synthesized N‑ and C‑terminal truncations or amino‑acid substitutions of buforin II and evaluated their antibacterial potency and cell‑penetration behavior. Deletion of the N‑terminal coil doubled activity, but further truncation reduced potency; removing the C‑terminal helix abolished activity; substituting proline with leucine markedly decreased activity and eliminated cell penetration, while analogs retaining the proline hinge entered cells without permeabilizing membranes, and the cell‑penetrating efficiency, which correlated linearly with α‑helical content, determined antimicrobial potency.

Abstract

Buforin II is a 21-aa potent antimicrobial peptide that forms, in a hydrophobic medium, an amphipathic structure consisting of an N-terminal random coil region (residues 1–4), an extended helical region (residues 5–10), a hinge (residue 11), and a C-terminal regular α-helical region (residues 12–21). To elucidate the structural features of buforin II that are required for its potent antimicrobial activity, we synthesized a series of N- and C-terminally truncated or amino acid-substituted synthetic buforin II analogs and examined their antimicrobial activity and mechanism of action. Deletion of the N-terminal random coil region increased the antibacterial activity ≈2-fold, but further N-terminal truncation yielded peptide analogs with progressively decreasing activity. Removal of four amino acids from the C-terminal end of buforin II resulted in a complete loss of antimicrobial activity. The substitution of leucine for the proline hinge decreased significantly the antimicrobial activity. Confocal fluorescence microscopic studies showed that buforin II analogs with a proline hinge penetrated the cell membrane without permeabilization and accumulated in the cytoplasm. However, removal of the proline hinge abrogated the ability of the peptide to enter cells, and buforin II analogs without a proline hinge localized on the cell surface, permeabilizing the cell membrane. In addition, the cell-penetrating efficiency of buforin II and its truncated analogs, which depended on the α-helical content of the peptides, correlated linearly with their antimicrobial potency. Our results demonstrate clearly that the proline hinge is responsible for the cell-penetrating ability of buforin II, and the cell-penetrating efficiency determines the antimicrobial potency of the peptide.