Defining the epitope region of a peptide from the Streptomyces coelicolor phosphoenolpyruvate:sugar phosphotransferase system able to bind to the enzyme I.
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 alpha-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.
Hurtado-Gómez
Estefanía
E
Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, 03202 Elche (Alicante), Spain.
Abián
Olga
O
Muñoz
F Javier
FJ
Hernáiz
María José
MJ
Velázquez-Campoy
Adrián
A
Neira
José L
JL
D001665:Binding Sites; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry*; D017852:Phosphotransferases (Nitrogenous Group Acceptor) / Q000737:chemistry*; D011485:Protein Binding; D048372:Streptomyces coelicolor / Q000201:enzymology*
D000939:Epitopes; D010455:Peptides; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D017852:Phosphotransferases (Nitrogenous Group Acceptor); C022360:phosphoenolpyruvate-protein phosphotransferase