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 transporter of Lactococcus lactis (DppA) was cloned under the control of the nisin promoter. Amplified expression ( approximately 200-fold increase) of the protein fused to a carboxyl-terminal six-histidine tag allowed the purification of DppA-(His)(6) by nickel-chelate affinity and anion-exchange chromatography. Ligand binding to DppA-(His)(6) elicited an electrophoretic mobility shift, a decrease in the intrinsic fluorescence, and a blue shift of the emission maximum. Each of these parameters detected conformational changes in the protein that reflect ligand binding, and these were used to determine the structural requirements of DppA-(His)(6) for binding peptides. The major features of peptide binding include (i) high affinity for di- and tripeptides, (ii) requirement of a free N-terminal alpha-amino group and an alpha-peptide bound contiguous with the N-terminal amino group, (iii) stereospecificity for L-isomers, and (iv) preference for dipeptides containing methionine or arginine, followed by hydrophobic tripeptides consisting of leucine or valine residues. Maximal binding affinity was detected at pH 6.0, and the K(d) for binding increased 1 order of magnitude for every unit increase in pH. This suggests that the ionization of protein residues (pK > 6.0) in or in close proximity to the binding site is critical in the binding mechanism.
Sanz
Y
Y
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, NL 9751 NN, Haren, The Netherlands.
Lanfermeijer
F C
FC
Konings
W N
WN
Poolman
B
B
D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001692:Biological Transport; D002352:Carrier Proteins; D004151:Dipeptides / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D013294:Lactococcus lactis / Q000737:chemistry*; D008024:Ligands; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D033902:Periplasmic Binding Proteins; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity; D013816:Thermodynamics; D017186:Titrimetry
D000596:Amino Acids; D001426:Bacterial Proteins; D002352:Carrier Proteins; D004151:Dipeptides; D029968:Escherichia coli Proteins; D008024:Ligands; D009842:Oligopeptides; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins; C066768:dppA protein, E coli