Maculatin 1.1

Maculatin 1.1 (Mac1) is an antimicrobial peptide (AMP) from an Australian tree frog and exhibits low micromolar activity against Gram-positive bacteria. The antimicrobial properties of Mac1 are linked to its disruption of bacterial lipid membranes, which has been studied extensively by in vitro nuclear magnetic resonance (NMR) spectroscopy and biophysical approaches. Although in vivo NMR has recently proven effective in probing peptide-lipid interplay in live bacterial cells, direct structural characterisation of AMPs has been prohibited by low sensitivity and overwhelming background noise. To overcome this issue, we report a recombinant expression protocol to produce isotopically enriched Mac1. We utilized a double-fusion construct to alleviate toxicity against the Escherichia coli host and generate the native N-free and C-amidated termini Mac1 peptide. The SUMO and intein tags allowed native N-terminus and C-terminal amidation, respectively, to be achieved in a one-pot reaction. The protocol yielded 0.1 mg/L of native, uniformly 15 N-labelled, Mac1, which possessed identical structure and activity to peptide obtained by solid-phase peptide synthesis.

The dorsal glands of Australian tree frogs from the Litoria species contain a diversity of antibiotic peptides that forms part of the defence system of the animal. Here, the antibiotic activity and structure of maculatin 1.1, a 21 amino acid peptide from Litoria genimaculata, are compared. The activity data on maculatin 1.1 and a series of its analogues imply that the mechanism of action of maculatin 1.1 involves binding to, and subsequent lysis of, the bacterial cell membrane. The structure of maculatin 1.1 was determined using NMR spectroscopy in a trifluoroethanol/water mixture and when incorporated into dodecylphosphocholine micelles. Under both conditions, the peptide adopts a very similar conformation, i.e. a helical structure with a central kink in the vicinity of Pro15. The kink allows the peptide to adopt a well-defined amphipathic conformation along its entire length. The similar structures determined under both solvent conditions imply that structures of membrane-interacting peptides in trifluoroethanol/water mixtures are representative of those adopted in a membrane environment, e.g. when incorporated into micelles. The synthetic Ala15 analogue of maculatin 1.1 has markedly reduced activity and its NMR-derived structure is a well-defined helix, which lacks the central kink and flexibility of the parent molecule. It is concluded that the kink is important for full biological activity of the peptide, probably because it allows maximum amphipathicity of the peptide to facilitate interaction with the membrane. The structure of maculatin 1.1 is compared with a related peptide, caerin 1.1 [Wong, H., Bowie, J.H. and Carver, J.A. (1997) Eur. J. Biochem. 247, 545-557], which has an additional central proline residue and enhanced central flexibility compared with maculatin 1.1. The role of central flexibility within antibiotic peptides in their interaction with bacterial membranes is discussed.

Maculatin 1.1 (Mac1) showed potent activity against Staphylococcus aureus with an MIC of 7 μM. The mode of action of Mac1 was investigated by combining assays with S. aureus cells and lipid vesicles mimicking their membrane composition. A change in Mac1 conformation was monitored by circular dichroism from random coil to ca. 70% α-helix structure in contact with vesicles. Electron micrographs of S. aureus incubated with Mac1 showed rough and rippled cell surfaces. An uptake of 65% of small (FD, 4 kDa [FD-4]) and 35% of large (RD, 40 kDa [RD-40]) fluorescent dextrans by S. aureus was observed by flow cytometry and indicate that Mac1 formed a pore of finite size. In model membranes with both dyes encapsulated together, the full release of FD-4 occurred, but only 40% of RD-40 was reached, supporting the flow cytometry results, and indicating a pore size between 1.4 and 4.5 nm. Finally, solid-state nuclear magnetic resonance showed formation of an isotropic phase signifying highly mobile lipids such as encountered in a toroidal pore structure. Overall, Mac1 is a promising antimicrobial peptide with the potent capacity to form pores in S. aureus membranes.