Structure-Function Analysis of the Two-Peptide Bacteriocin Plantaricin EF

Area of Science:

• Microbiology
• Structural Biology
• Biochemistry

Background:

• Plantaricin EF is a two-peptide bacteriocin requiring complementary PlnE and PlnF peptides for activity.
• Previous studies determined individual peptide structures but not their interaction or overall bacteriocin structure.
• GxxxG motifs are known to mediate helix-helix interactions in membrane proteins and are present in all identified two-peptide bacteriocins.

Purpose of the Study:

• To elucidate the structure and interaction mechanism of the two-peptide bacteriocin Plantaricin EF.
• To identify the role of GxxxG and GxxxG-like motifs in the antimicrobial activity and inter-peptide interactions of PlnE and PlnF.
• To determine the relative orientation of PlnE and PlnF within target cell membranes.

Main Methods:

• Site-directed mutagenesis of GxxxG and GxxxG-like motifs in PlnE and PlnF.
• Substitution of aromatic amino acids (Tyr and Trp) in PlnE and PlnF.
• Construction of fusion polypeptides to investigate peptide orientation.
• Atomistic molecular dynamics simulations of a structural model.

Main Results:

• Fusion polypeptides indicated an antiparallel interaction between PlnE and PlnF.
• The C-terminus of PlnE and N-terminus of PlnF face the outer membrane, while the N-terminus of PlnE and C-terminus of PlnF are on the inner side.
• Mutations in the G5xxxG9 motif of PlnE and S26xxxG30 motif of PlnF impaired antimicrobial activity, suggesting their role in helix-helix interactions.
• Molecular dynamics simulations confirmed the stability and orientation of the proposed structure, revealing additional stabilizing interactions.

Conclusions:

• Plantaricin EF's structure involves an antiparallel arrangement of PlnE and PlnF peptides within the membrane.
• Specific GxxxG motifs (PlnE G5xxxG9 and PlnF S26xxxG30) are crucial for mediating helix-helix interactions and overall antimicrobial function.
• The study provides a stable structural model for Plantaricin EF, enhancing understanding of two-peptide bacteriocin mechanisms.