TY - JOUR
T1 - Structure-Function Analysis of the Two-Peptide Bacteriocin Plantaricin EF
AU - Ekblad, Bie
AU - Kyriakou, Panagiota K.
AU - Oppegård, Camilla
AU - Nissen-Meyer, Jon
AU - Kaznessis, Yiannis
AU - Kristiansen, Per Eugen
N1 - Publisher Copyright:
© 2016 American Chemical Society.
PY - 2016/9/13
Y1 - 2016/9/13
N2 - Plantaricin EF is a two-peptide bacteriocin that depends on the complementary action of two different peptides (PlnE and PlnF) to function. The structures of the individual peptides have previously been analyzed by nuclear magnetic resonance spectroscopy (Fimland, N. et al. (2008), Biochim. Biophys. Acta 1784, 1711-1719), but the bacteriocin structure and how the two peptides interact have not been determined. All two-peptide bacteriocins identified so far contain GxxxG motifs. These motifs, together with GxxxG-like motifs, are known to mediate helix-helix interactions in membrane proteins. We have mutated all GxxxG and GxxxG-like motifs in PlnE and PlnF in order to determine if any of these motifs are important for antimicrobial activity and thus possibly for interactions between PlnE and PlnF. Moreover, the aromatic amino acids Tyr and Trp in PlnE and PlnF were substituted, and four fusion polypeptides were constructed in order to investigate the relative orientation of PlnE and PlnF in target cell membranes. The results obtained with the fusion polypeptides indicate that PlnE and PlnF interact in an antiparallel manner and that the C-terminus of PlnE and N-terminus of PlnF are on the outer part of target cell membranes and the N-terminus of PlnE and C-terminus of PlnF are on the inner part. The preference for an aromatic residue at position 6 in PlnE suggests a positioning of this residue in or near the membrane interface on the cells inside. Mutations in the GxxxG motifs indicate that the G5xxxG9 motif in PlnE and the S26xxxG30 motif in PlnF are involved in helix-helix interactions. Atomistic molecular dynamics simulation of a structural model consistent with the results confirmed the stability of the structure and its orientation in membranes. The simulation approved the anticipated interactions and revealed additional interactions that further increase the stability of the proposed structure.
AB - Plantaricin EF is a two-peptide bacteriocin that depends on the complementary action of two different peptides (PlnE and PlnF) to function. The structures of the individual peptides have previously been analyzed by nuclear magnetic resonance spectroscopy (Fimland, N. et al. (2008), Biochim. Biophys. Acta 1784, 1711-1719), but the bacteriocin structure and how the two peptides interact have not been determined. All two-peptide bacteriocins identified so far contain GxxxG motifs. These motifs, together with GxxxG-like motifs, are known to mediate helix-helix interactions in membrane proteins. We have mutated all GxxxG and GxxxG-like motifs in PlnE and PlnF in order to determine if any of these motifs are important for antimicrobial activity and thus possibly for interactions between PlnE and PlnF. Moreover, the aromatic amino acids Tyr and Trp in PlnE and PlnF were substituted, and four fusion polypeptides were constructed in order to investigate the relative orientation of PlnE and PlnF in target cell membranes. The results obtained with the fusion polypeptides indicate that PlnE and PlnF interact in an antiparallel manner and that the C-terminus of PlnE and N-terminus of PlnF are on the outer part of target cell membranes and the N-terminus of PlnE and C-terminus of PlnF are on the inner part. The preference for an aromatic residue at position 6 in PlnE suggests a positioning of this residue in or near the membrane interface on the cells inside. Mutations in the GxxxG motifs indicate that the G5xxxG9 motif in PlnE and the S26xxxG30 motif in PlnF are involved in helix-helix interactions. Atomistic molecular dynamics simulation of a structural model consistent with the results confirmed the stability of the structure and its orientation in membranes. The simulation approved the anticipated interactions and revealed additional interactions that further increase the stability of the proposed structure.
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U2 - 10.1021/acs.biochem.6b00588
DO - 10.1021/acs.biochem.6b00588
M3 - Article
C2 - 27538436
AN - SCOPUS:84987667447
SN - 0006-2960
VL - 55
SP - 5106
EP - 5116
JO - Biochemistry
JF - Biochemistry
IS - 36
ER -