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Substrate specificity of the periplasmic dipeptide-binding protein from Escherichia coli: experimental basis for the design of peptide prodrugs

School of Biological Sciences, University of Wales, Bangor LL57 2UW, UK¹

Author for correspondence: John W. Payne. Tel: +44 1248 382349. Fax: +44 1248 370731. e-mail: j.w.payne{at}bangor.ac.uk

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 -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.

Keywords: Escherichia coli, dipeptide transport, peptide prodrugs, periplasmic binding protein, antibacterial peptides

Abbreviations: Dpp, dipeptide permease; Opp, oligopeptide permease; RPC, reverse-phase chromatography; TFA, trifluoroacetic acid; Tpp, tripeptide permease

^a Present address: Cortecs plc, Newtech Square, Deeside Industrial Park, Flintshire CH5 2NT, UK.

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