Kinetics and structural requirements for the binding protein of the di-tripeptide transport system of Lactococcus lactis

The gene (dppA) encoding the binding protein of the di-tripeptide ABC trans porter of Lactococcus lactis (DppA) was cloned under the control of the nis in promoter. Amplified expression (approximate to 200-fold increase) of the protein fused to a carboxyl-terminal six-histidine tag allowed the purific ation of DppA-(His)(6) by nickel-chelate affinity and anion-exchange chroma tography. Ligand binding to DppA-(His)(6) elicited an electrophoretic mobil ity shift, a decrease in the intrinsic fluorescence, and a blue shift of th e emission maximum. Each of these parameters detected conformational change s in the protein that reflect ligand binding, and these were used to determ ine the structural requirements of DppA-(His)(6) for binding peptides. The major features of peptide binding include (i) high affinity for di- and tri peptides, (ii) requirement of a free N-terminal alpha-amino group and an al pha-peptide bound contiguous with the N-terminal amino group, (iii) stereos pecificity for L-isomers, and (iv) preference for dipeptides containing met hionine or arginine, followed by hydrophobic tripeptides consisting of leuc ine of valine residues. Maximal binding affinity was detected at pH 6.0, an d the K-d for binding increased I order of magnitude for every unit increas e in pH. This suggests that the ionization of protein residues (pK > 6.0) i n or in close proximity to the binding site is critical in the binding mech anism.