Electrostatics-Based Computational Design and Experimental Analysis of Buforin II Antimicrobial Peptide Variants with Increased DNA Affinities

Abstract

Antimicrobial peptides (AMPs) are promising alternatives to traditional antibiotics in the treatment of bacterial infections in part due to their targeting of generic bacterial structures that make it more difficult to develop drug resistance. In this study, we introduce and implement a design workflow to develop more potent AMPs by improving their electrostatic interactions with DNA, which is a putative intracellular target. Using the existing membrane-translocating AMP buforin II (BF2) as a starting point, we use a computational workflow that integrates electrostatic charge optimization, continuum electrostatics, and molecular dynamics simulations to suggest peptide positions at which a neutral BF2 residue could be substituted with arginine to increase DNA-binding affinity either significantly or minimally, with the latter choice done to determine whether AMP binding affinity depends on charge distribution and not just overall monopole. Our analyses predicted that T1R and L8R BF2 variants would yield substantial and minimal increases in DNA-binding affinity, respectively. These predictions were validated with experimental peptide-DNA binding assays with additional computational analyses providing structural insights. Additionally, experimental measurements of antimicrobial potency showed that a design to increase DNA binding can also yield greater potency. As a whole, this study takes initial steps to support the idea that (i) a design strategy aimed to increase AMP binding affinity to DNA by focusing only on electrostatic interactions can improve AMP potency and (ii) the effect on DNA binding of increasing the overall peptide monopole via arginine substitution depends on the position of the substitution. More broadly, this design strategy is a novel way to increase the potency of other membrane-translocating AMPs that target nucleic acids.

Photobuforin II, a fluorescent photoswitchable peptide

“…Literature values of 50 % binding for buforin II have determined the value to be around 11 μM. [34] Thus, photobuforin II appeared to maintain much of the wild type peptide affinity for DNA binding. The binding of DNA to buforin II most likely involves electrostatic interactions between the phosphate groups of the DNA and basic residues of the peptide.…”

Bioactive Peptides and Protein Hydrolysates Used in Meat and Meat Products’ Preservation─A Review

“…For example, buforin II, an antimicrobial peptide found in the skin of Asian toads of the genus Bufo with antimicrobial activity against fungi, viruses, and a wide range of Gram-positive and Gram-negative bacteria, may attach to DNA and RNA and permeate into cells without rupturing the cell membrane. 71 Antimicrobial protein hydrolysates and peptides have been identified from natural sources such as BAL, insects, and plants, specifically grains like wheat and legumes. 33,72,73 Among insects, Order Hymenoptera (ants, bees, and wasps) is the most studied regarding antimicrobial peptides.…”

“…Some antimicrobial peptides can kill bacteria by blocking vital cellular processes, including DNA replication and protein synthesis, as opposed to directly interacting with the membrane. For example, buforin II, an antimicrobial peptide found in the skin of Asian toads of the genus Bufo with antimicrobial activity against fungi, viruses, and a wide range of Gram-positive and Gram-negative bacteria, may attach to DNA and RNA and permeate into cells without rupturing the cell membrane …”