Truncated Equinin B Variants Reveal the Sequence Determinants of Antimicrobial Selectivity

Staropoli, Mariele; Schwaiger, Theresa; Tuzlak, Jasmina; Biba, Renata; Petrowitsch, Lukas; Fessler, Johannes; Roje, Marin; Cammarata, Matteo; Malanovic, Nermina; Jakas, Andreja (2026) Truncated Equinin B Variants Reveal the Sequence Determinants of Antimicrobial Selectivity. Marine Drugs, 24 (1). ISSN 1660-3397

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Abstract

Equinin B (GQCQRKCLGHCSKKCPKHPQCRKRCIRRCFGYCL), a marine peptide from Actinia equina exhibits antibacterial activity against both Gram-positive and Gram-negative bacteria. To identify a smaller active region and explore tunable properties, three peptide fragments were synthesized: GQCQRKCLGHCS (EB1), KKCPKHPQCRK (EB2), and RCIRRCFGYCL (EB3), yielding peptides with key AMP-like properties, including the most positively charged and most hydrophobic regions. Only the 11-residue C-terminal fragment showed selective activity against Gram-positive bacteria, including Staphylococcus epidermidis, Bacillus subtilis, and Enterococcus hirae, while remaining inactive against Escherichia coli. Peptide modifications, achieved by replacing cysteine residues with arginine, generally did not enhance activity, but in the C-terminal fragment EB3 they reduced hemolytic activity and increased bacterial specificity. Membrane depolarization assays confirmed that the unmodified fragment EB3 strongly compromises bacterial membranes, whereas the modified variant showed minimal depolarization, highlighting its markedly reduced membrane-perturbing potential. In silico modelling revealed that the EB3 can adopt multiple membrane-disruption modes, from transient shallow pores to carpet-like mechanisms, while the cysteine-to-arginine variant interacts mainly via partial insertion anchored by arginine residues. Phenylalanine appears to interact with the membrane, and reducing hydrophobicity by its removal abolished antibacterial activity. These findings highlight the 11-residue C-terminal fragment as a tunable, membrane-targeting motif with mechanistic novelty, offering a blueprint for developing safer, selective antimicrobial peptides with reduced cytotoxicity.

Item Type:	Article
Uncontrolled Keywords:	marine peptides; solid phase peptide synthesis; structure-function relationship; membrane permeability; peptide specificity; peptide–membrane interactions; Gram-positive bacteria
Subjects:	NATURAL SCIENCES > Chemistry BIOMEDICINE AND HEALTHCARE > Pharmacy
Divisions:	Division of Molecular Medicine Division of Organic Chemistry and Biochemistry
Projects:	Project title Project leader Project code Project type Antimikrobni peptidi temeljeni na prirodnim morskim antimikrobicima: Dizajn i studija načina djelovanja-AMPNatMarAM Andreja Jakas IP-2024-05-1216 HRZZ Metaproteomika patvorenih namirnica-MetaPatvor Mario Cindrić; Ivana Varenina NPOO.C3.2.R3-I1.04.0122 EK Znanstveni centar izvrsnosti za bioprospecting mora-BioProCro Rozelinda Čož-Rakovac; Marin Roje N/A MZOS
Depositing User:	Ivana Vuglec
Date Deposited:	02 Jun 2026 12:25
URI:	https://fulir.irb.hr:/id/eprint/12004
DOI:	10.3390/md24010046

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