Buforin III Analogs Bind to DNA and Actin and Inhibit Bacterial Growth

Abstract

Buforin is a cationic antimicrobial peptide (AMP) from the stomach of toads. Buforin II is a derivative of this naturally occurring peptide. Buforin IIB is a synthetic analog of buforin II containing a model α-helical sequence (3xRLLR) at the C-terminus. To further increase the antimicrobial activity and decrease toxicity to eukaryotic cells, new derivatives (buforin III analogs) were designed by substituting amino acids in the buforin IIB sequence. In this work, the antimicrobial activity and the actin-and DNA-binding characteristics of buforin IIIB (RVVRQWPIGRVVRRVVRRVVR) and the newly synthetized buforin IIIE (RLLLRQWPIGRLLRRLLRRLLR) were studied. The antimicrobial activity of buforin IIIB (measured against E. coli and E. faecalis) was significantly greater than that of buforin IIIE, while both peptides were nontoxic to macrophages at the minimal concentrations required to inhibit microbial growth. Actin, which inhibited the antimicrobial activity of the two buforin III analogs, was bundled by both peptides; however, less buforin IIIE than buforin IIIB was needed for bundling. Higher levels of NaCl were needed to unbundle actin bundled by buforin IIIE than actin bundled by buforin IIIB, which indicates that buforin IIIE binds more strongly to actin than buforin IIIB. Actin bundled by either peptide was dissociated with the same concentration of DNA; however, buforin IIIE bound more strongly to DNA than buforin IIIB. These results contribute to the understanding of the antimicrobial mechanism of cationic AMPs in general and histone-derived peptides in particular.

Interaction of Extracellular Histones with DNA and Actin Filaments

“…As the number positive charges in H1 is significantly higher than in the other histones it is possible that to the relatively low ionic strength sensitivity of H1 is the results of both the higher electrostatic and hydrophobic interactions of this histone than the other extracellular histones with the actin filaments. DNA was shown to dissociate histone [14] and histone derivative (buforin) [24] bundled F-actin, because the strongly negatively charged DNA competes for the positively charged histones with the negatively charged F-actin. We studied the effect of DNA on the H2A, H2B, H3.1, H4 and H1 histone induced F-actin bundles with low speed centrifugation ( Figure 5), as the histone-DNA complexes, unlike the histone-F-actin complexes, are not sedimented with low speed centrifugation.…”

“…DNase1 is a G-actin binding protein. It binds very tightly to the D-loop of G-actin and forms co-crystals with actin monomers [24] and depolymerizes F-actin by binding to the protomer at the filaments ends. DNase 1 depolymerizes Factin filaments to G-actin by dissociating actin monomers from the end of the filaments [25].…”