Structure-Function Analysis of Tritrypticin, an Antibacterial Peptide of Innate Immune Origin

Sushma Nagpal,Vibha Gupta,Kanwal J Kaur,Dinakar M Salunke

doi:10.1074/jbc.274.33.23296

Sushma Nagpal, Vibha Gupta + Show 2 more

Open Access

https://doi.org/10.1074/jbc.274.33.23296

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Abstract

The structural requirements for the antibacterial activity of a pseudosymmetric 13-residue peptide, tritrypticin, were analyzed by combining pattern recognition in protein structures, the structure-activity knowledge-base, and circular dichroism. The structure-activity analysis, based on various deletion analogs, led to the identification of two minimal functional peptides, which by themselves exhibit adequate antibacterial activity against Escherichia coli and Salmonella typhimurium. The common features between these two peptides are that they both share an aromatic-proline-aromatic (ArProAr) sequence motif, and their sequences are retro with respect to one another. The pattern searches in protein structure data base using the ArProAr motif led to the identification of two distinct conformational clusters, namely polyproline type II and beta-turn, which correspond to the observed solution structures of the two minimal functional analogs. The role of different residues in structure and function of tritrypticin was delineated by analyzing antibacterial activity and circular dichroism spectra of various designed analogs. Three main results arise from this study. First, the ArProAr sequence motif in proteins has definitive conformational features associated with it. Second, the two minimal bioactive domains of tritrypticin have entirely different structures while having equivalent activities. Third, tritrypticin has a beta-turn conformation in solution, but the functionally relevant conformation of this gene-encoded peptide antibiotic may be an extended polyproline type II.

Highlights

The survival of all living organisms necessitates a rapid and effective host defense against invading pathogens
The higher organisms that have arrived much later in a world inhabited by prokaryotes developed many host defense mechanisms, including gene-encoded antimicrobial peptides to face the challenge of these pathogens [1, 2]
The dose-dependent increase in the antibacterial activity of SN13 was evident in both the cases, it is slightly more active against E. coli than S. typhimurium

Summary

EXPERIMENTAL PROCEDURES

Materials—4-Hydroxymethyl phenoxymethyl polystyrene resin, solvents, and reagents used for synthesis were supplied by Applied Biosystems Inc. Fmoc amino acid derivatives were procured from Novabiochem and Bachem Feinchemikalein AG. Trifluoroacetic acid, 1,2-ethanedithiol, and thioanisole for cleavage were procured from Sigma. High performance liquid chromatography grade acetonitrile was obtained from Merck. Agarose I (Biotechnology grade) was obtained from Amresco, and tryptic soy broth (TSB) was from Himedia Laboratories Pvt. Ltd. Sterilized round Petri plates were purchased from Tarsons (Calcutta, India). The peptides were cleaved from the resin by treatment with trifluoroacetic acid/thioanisole/phenol/ water/1,2-ethanedithiol in ratio as recommended by Applied Biosystems Inc. The crude peptides were purified using C-18 column (Deltapak-100Å, 15 ␮m spherical, 19 ϫ 300 mm, Waters), and peptide purity was verified using C-18 analytical column (Deltapak-300Å, 15 ␮m, spherical, 7.8 ϫ 300 mm, Waters). Elution of the peptides was accomplished with a linear gradient from 15 to 80% acetonitrile con-

The abbreviations used are

RESULTS

DISCUSSION