Substrate specificity of the periplasmic dipeptide-binding protein from Escherichia coli: experimental basis for the design of peptide prodrugs.
Pure dipeptide-binding protein (DppA) from Escherichia coli was studied in a filter binding assay to determine its binding specificity. A substrate:DppA stoichiometry of 1:1 was found with both [14C]AlaAla and Ala[14C]Phe. Surprisingly, substrate binding did not vary over the pH range pH 3-9.5. Different dipeptides yielded liganded protein with various pI values, implying that DppA can undergo subtly different conformational changes to accommodate different substrates. Using [125I]Tyr-peptides as substrates in competition assays, the relative binding affinities for a range of dipeptides were found to parallel their overall transport rates into E. coli through the dipeptide permease (Dpp), showing that DppA alone controls the specificity of Dpp. With a series of substituted glycyl peptides, binding affinity was progressively enhanced by alkylation (with methyl to butyl) of the N-terminal alpha-amino group. Thus, results from this approach provide an essential experimental basis, which complements the information from the crystal structure of DppA, for the design of peptidomimetic antibacterials targeted for transport through Dpp.
Smith
M W
MW
School of Biological Sciences, University of Wales, Bangor, UK.
Tyreman
D R
DR
Payne
G M
GM
Marshall
N J
NJ
Payne
J W
JW
D000597:Amino Acids, Branched-Chain / Q000378:metabolism; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001667:Binding, Competitive; D001692:Biological Transport; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004151:Dipeptides / Q000378:metabolism; D004337:Drug Carriers; D015195:Drug Design; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism* / Q000627:therapeutic use; D033902:Periplasmic Binding Proteins; D011355:Prodrugs / Q000378:metabolism*; D011487:Protein Conformation; D020033:Protein Isoforms; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D000597:Amino Acids, Branched-Chain; D001426:Bacterial Proteins; D002352:Carrier Proteins; D004151:Dipeptides; D004337:Drug Carriers; D029968:Escherichia coli Proteins; D009842:Oligopeptides; D033902:Periplasmic Binding Proteins; D011355:Prodrugs; D020033:Protein Isoforms; C066768:dppA protein, E coli