Antimicrobial peptide ROAD-1 triggers phase change in local membrane environment to execute its activity.

Emergence of antibiotic-resistant pathogens has paved way for development of newer class of drugs that would not be susceptible to resistance. Antimicrobial peptides such as defensins that target the microbial membrane are promising candidates. ROAD-1 is an alpha-defensin present in the oral cavity of rhesus macaque and shares very high sequence similarity to human enteric defensin 5. In this study we have performed microsecond long all atom molecular dynamic simulations to understand the mechanism of action of ROAD-1. We find that ROAD-1 is able to adopt an energetically stable conformation predominantly stabilized by electrostatic interactions only in presence of bacterial membranes. In mammalian membrane even though it gets absorbed onto the bilayer, it is unable to adopt an equilibrium conformation. Binding of ROAD-1 to bilayer induces clustering of POPG molecules up to 15 Å around the peptide. POPG molecules show higher order parameters than the neighboring POPE implying coexistence of different phases. Analysis of binding free energy of ROAD-1-membrane complex indicates Arg1, Arg2, Arg7, and Arg25 to play key role in its antimicrobial activity. Unlike its homolog HD5, ROAD-1 is not observed to form a dimer. Our study gives insight into the membrane-bound conformation of ROAD-1 and its mechanism of action that can aid in designing defensin-based therapeutics. Graphical abstract Antimicrobial peptide ROAD-1 adopts a different membrane-bound conformation as compared with HD5 even though they belong to the same family implying a different mechanism of action.