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

¹ Universidad Miguel Hernandez
² Universidad de Zaragoza
³ Universidad Complutense de Madrid
⁴ University of Miguel Hernandez

^* To whom correspondence should be addressed. E-mail: jlneira{at}umh.es.

Submitted on November 27, 2007
Revised on January 10, 2008
Accepted on 11 April 2008

	Abstract

The bacterial phosphoenolpyruvate (PEP):sugar phosphotransferase system (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 histidine-phosphocarrier protein, 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 (His15) and the first -helix of HPr of Streptomyces coelicolor, HPr^sc. By using fluorescence and circular dichroism, we firstly 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 Leu14, His15, Ile21 and Val23. 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.

Key Words: Binding, Inhibition, NMR, Peptide, Titration calorimetry