Defining the epitope region of a peptide from the Streptomyces coelicolor phosphoenolpyruvate:sugar phosphotransferase system able to bind to the enzyme I

Abstract

The bacterial PEP:sugar PTS consists of a cascade of several proteins involved in the uptake and phosphorylation of carbohydrates, and in signal transduction pathways. Its uniqueness in bacteria makes the PTS a target for new antibacterial drugs. These drugs can be obtained from peptides or protein fragments able to interfere with the first reaction of the protein cascade: the phosphorylation of the HPr by the first enzyme, the so-called enzyme EI. To that end, we designed a peptide, HPr9-30, spanning residues 9 to 30 of the intact HPr protein, containing the active site histidine (His-15) and the first α-helix of HPr of Streptomyces coelicolor, HPrsc. By using fluorescence and circular dichroism, we first determined qualitatively that HPrsc and HPr9-30 did bind to EIsc, the enzyme EI from S. coelicolor. Then, we determined quantitatively the binding affinities of HPr9-30 and HPrsc for EIsc by using ITC and STD-NMR. The STD-NMR experiments indicate that the epitope region of HPr9-30 was formed by residues Leu-14, His-15, Ile-21, and Val-23. The binding reaction between EIsc and HPrsc is enthalpy driven and in other species is entropy driven; further, the affinity of HPr sc for EIsc was smaller than in other species. However, the affinity of HPr9-30 for EIsc was only moderately lower than that of EIsc for HPrsc, suggesting that this peptide could be considered a promising hit compound for designing new inhibitors against the PTS. © 2008 by the Biophysical Society.