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, HPr 9–30, 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, HPr sc. By using fluorescence and circular dichroism, we first determined qualitatively that HPr sc and HPr 9–30 did bind to EI sc, the enzyme EI from S. coelicolor. Then, we determined quantitatively the binding affinities of HPr 9–30 and HPr sc for EI sc by using ITC and STD-NMR. The STD-NMR experiments indicate that the epitope region of HPr 9–30 was formed by residues Leu-14, His-15, Ile-21, and Val-23. The binding reaction between EI sc and HPr sc is enthalpy driven and in other species is entropy driven; further, the affinity of HPr sc for EI sc was smaller than in other species. However, the affinity of HPr 9–30 for EI sc was only moderately lower than that of EI sc for HPr sc, suggesting that this peptide could be considered a promising hit compound for designing new inhibitors against the PTS.