Combinatorial peptide libraries reveal the ligand-binding mechanism of theoligopeptide receptor OppA of Lactococcus lactis

The oligopeptide transport system (Opp) of Lactococcus lactis has the uniqu e capacity to mediate the transport of peptides from 4 up to at least 18 re sidues. The substrate specificity of this binding protein-dependent ATP-bin ding cassette transporter is determined mainly by the receptor protein OppA . To study the specificity and ligand-binding mechanism of OppA, the follow ing strategy was used: (i) OppA was purified and anchored via the lipid moi ety to the surface of liposomes; (ii) the proteoliposomes were used in a ra pid filtration-based binding assay with radiolabeled nonameric bradykinin a s a reporter peptide; and (iii) combinatorial peptide libraries were used t o 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 fo r nonameric peptides is not equally distributed over the whole peptide, bec ause 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, bu t overall hydrophobic residues are favored-whereas glycine, proline, and ne gatively charged residues lower the binding affinity. The data indicate tha t not only the first six residues (enclosed by the protein) but also the C- terminal three residues interact in a nonopportunistic: manner with (the su rface of) OppA, This binding mechanism is different from the one generally accepted for receptors of ATP-binding cassette-transporter systems.