Mutational analysis of residues in the helical region of the class IIa bacteriocin pediocin PA-1.
A 15-mer fragment that is derived from the helical region in the C-terminal half of pediocin PA-1 inhibited the activity of pediocin PA-1. Of 13 other pediocin-like (hybrid) bacteriocins, only the hybrid bacteriocin Sak/Ped was markedly inhibited by the 15-mer fragment. Sak/Ped was the only one of these bacteriocins that had a sequence (in the C-terminal helix-containing half) identical to that of the 15-mer fragment, indicating that the fragment inhibits pediocin-like bacteriocins in a sequence-dependent manner. By replacing (one at a time) all 15 residues in the fragment with Ala or Leu, five residues (K1, A2, T4, N8, and A15) were identified as being especially important for the inhibitory action of the fragment. The results suggest that the corresponding residues (K20, A21, T23, N27, and A34, respectively) in pediocin PA-1 might be involved in interactions between pediocin PA-1 and its receptor. To characterize the environment surrounding these five residues when pediocin PA-1 interacts with target cells, these residues were replaced (one at a time) with a hydrophobic large (Leu) residue, a hydrophilic charged (Asp or Arg) residue, and a small (Ala or Gly) residue. The results revealed that residues A21 and A34 are in a spatially constrained environment, since the replacement with a small (Gly) residue was the only substitution that did not markedly reduce the bacteriocin activity. The positive charge in K20 and the polar amide group in N27 appeared to interact with electronegative groups, since the replacement of these two residues with a positive (Arg) residue was well tolerated, while replacement with a negative (Asp) residue was detrimental to the bacteriocin activity. K20 was in a less constrained environment than N27, since the replacement of K20 with a large hydrophobic (Leu) residue was tolerated fairly well and to a greater extent than N27. T23 seemed to be in an environment that was not restricted with respect to size, polarity, and charge, since replacements with large (Leu) and small (Ala) hydrophobic residues and a hydrophilic negative (Asp) residue were tolerated fairly well (2- to 6-fold reduction in activity). Moreover, the replacement of T23 with a large positive (Arg) residue resulted in wild-type or better-than-wild-type activity.
Department of Molecular Biosciences, University of Oslo, P.O. Box 1041 Blindern, 0316 Oslo, Norway. h.s.haugen@imbv.uio.no
Helén Sophie
HS
Haugen
Gunnar
G
Fimland
Jon
J
Nissen-Meyer
D000595:Amino Acid Sequence; D001430:Bacteriocins / Q000737:chemistry* / Q000235:genetics* / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D000070017:Pediocins; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D001430:Bacteriocins; D000070017:Pediocins; C074288:pediocin PA-1
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