Combinatorial peptide libraries reveal the ligand-binding mechanism of the oligopeptide receptor OppA of Lactococcus lactis.
The oligopeptide transport system (Opp) of Lactococcus lactis has the unique capacity to mediate the transport of peptides from 4 up to at least 18 residues. The substrate specificity of this binding protein-dependent ATP-binding cassette transporter is determined mainly by the receptor protein OppA. To study the specificity and ligand-binding mechanism of OppA, the following strategy was used: (i) OppA was purified and anchored via the lipid moiety to the surface of liposomes; (ii) the proteoliposomes were used in a rapid filtration-based binding assay with radiolabeled nonameric bradykinin as a reporter peptide; and (iii) combinatorial peptide libraries were used to determine the specificity and selectivity of OppA. The studies show that (i) OppA is able to bind peptides up to at least 35 residues, but there is a clear optimum in affinity for nonameric peptides; (ii) the specificity for nonameric peptides is not equally distributed over the whole peptide, because positions 4, 5, and 6 in the binding site are more selective; and (iii) the differences in affinity for given side chains is relatively small, but overall hydrophobic residues are favored-whereas glycine, proline, and negatively charged residues lower the binding affinity. The data indicate that not only the first six residues (enclosed by the protein) but also the C-terminal three residues interact in a nonopportunistic manner with (the surface of) OppA. This binding mechanism is different from the one generally accepted for receptors of ATP-binding cassette-transporter systems.
Detmers
F J
FJ
Departments of Microbiology, and Biochemistry, Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands.
Lanfermeijer
F C
FC
Abele
R
R
Jack
R W
RW
Tampe
R
R
Konings
W N
WN
Poolman
B
B
D000596:Amino Acids / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D001920:Bradykinin / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D013294:Lactococcus lactis / Q000378:metabolism*; D008024:Ligands; D008074:Lipoproteins / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism*; D019151:Peptide Library; D011869:Radioligand Assay
D000596:Amino Acids; D001426:Bacterial Proteins; D002352:Carrier Proteins; D008024:Ligands; D008074:Lipoproteins; D009842:Oligopeptides; D019151:Peptide Library; C046506:oligopeptide-binding protein, bacteria; D001920:Bradykinin