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Role of proline, cysteine and a disulphide bridge in the structure and activity of the anti-microbial peptide gaegurin 5.

ABSTRACT: Gaegurin 5 (GGN5) is a cationic 24-residue anti-microbial peptide isolated from the skin of a Korean frog, Rana rugosa. It contains a central proline residue and an intra-residue disulphide bridge in its C-terminus, which are common to the anti-microbial peptides found in Ranidae. We determined the solution structure of GGN5 bound to SDS micelles for the first time and investigated the role of proline, cysteine and a disulphide bridge on the structure and activity of GGN5. GGN5 adopts an amphipathic alpha-helical structure spanning residues 3-20 kinked around Pro-14, which allows the hydrophobic residues to reside in the concave helical region, and a disulphide-bridged loop-like conformation in its C-terminus. By replacement of proline with alanine (PAGGN5), a straight and rigid helix was formed in the central region and was more stable than the kinked helix. Reduction of a disulphide bridge in the C-terminus (GGN5SH) maintained the loosely ordered loop-like conformation, while the replacement of two cysteines with serines (CSGGN5) caused the C-terminal conformation to be completely disordered. The magnitude of anti-microbial activity of the peptides was closely related to their helical stability in the order PAGGN5>GGN5>GGN5SH>CSGGN5, suggesting that the helical stability of the peptides is important for anti-microbial activity. On the other hand, the significant increase of haemolytic activity of PAGGN5 implies that a helical kink of GGN5 could be involved in the selectivity of target cells. The location of GGN5 and PAGGN5, analysed using paramagnetic probes, was mainly at the surface of SDS micelles, although the location of the N-terminal region was slightly different between them.

SUBMITTER: Park SH

PROVIDER: S-EPMC1222972 | biostudies-other | 2002 Nov

REPOSITORIES: biostudies-other

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Project description:YejABEF is an ATP-binding cassette transporter that is implicated in the sensitivity of Escherichia coli to anti-microbial peptides, the best-characterized example being microcin C, a peptide-nucleotide antibiotic that targets aspartyl-tRNA synthetase. Here the structure of the extracellular solute binding protein, YejA, has been determined, revealing an oligopeptide-binding protein fold enclosing a ligand-binding pocket larger than those of other peptide-binding proteins of known structure. Prominent electron density in this cavity defines an undecapeptide sequence LGEPRYAFNFN, an observation that is confirmed by mass spectrometry. In the structure, the peptide interactions with the protein are mediated by main chain hydrogen bonds with the exception of Arg5 whose guanidinium side chain makes a set of defining polar interactions with four YejA residues. More detailed characterization of purified recombinant YejA, by a combination of ESI and MALDI-mass spectrometry as well as thermal shift assays, reveals a set of YejA complexes containing overlapping peptides 10-19 residues in length. All contain the sequence LGEPRYAFN. Curiously, these peptides correspond to residues 8-26 of the mature YejA protein, which belong to a unique N-terminal extension that distinguishes YejA from other cluster C oligopeptide binding proteins of known structure. This 35-residue extension is well-ordered and packs across the surface of the protein. The undecapeptide ligand occupies only a fraction of the enclosed pocket volume suggesting the possibility that much larger peptides or peptide conjugates could be accommodated, though thermal shift assays of YejA binding to antimicrobial peptides and peptides unrelated to LGEPRYAFNFN have not provided evidence of binding. While the physiological significance of this 'auto-binding' is not clear, the experimental data suggest that it is not an artefact of the crystallization process and that it may have a function in the sensing of periplasmic or membrane stress.

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Role of proline, cysteine and a disulphide bridge in the structure and activity of the anti-microbial peptide gaegurin 5.

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