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 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.

The Histidine-Phosphocarrier Protein of the Phosphoenolpyruvate: Sugar Phosphotransferase System of Bacillus sphaericus Self-Associates

“…6), unless we have experimental evidence, we cannot rule out fully this second explanation. Next, the higher value could be due to the presence of the self-association equilibrium involving the HPr bs ; for instance, we have observed that dimerization of the intact EI sc affects its binding to HPr sc [62]. And finally, we have shown that peptides [20], which do not mimic the natural target of EIN sc -HPr sc interface (instead, they were obtained by phage display and combinatorial techniques), bound stronger to EIN sc than peptides derived from PTS proteins of the same species.…”

Residual Helicity at the Active Site of the Histidine Phosphocarrier, HPr, Modulates Binding Affinity to Its Natural Partners

“…We preferred using the isolated mutant peptides to study the effect of helicity on binding, instead of the entire mutated HPr, since we showed previously that the isolated wild-type peptide of HPr ( Table 3 ) had a similar affinity for EIN and Rsd to that of the intact HPr for the same proteins [ 24 , 28 , 31 ]. By using the designed peptides ( Table 1 ), we avoided any other possible interactions that could have the mutated residues with the rest of HPr.…”

“…We have carried out an extensive description of the structures and conformational stabilities of HPr sc (O50515) and EI (EI sc ) (Q9KZP1) to understand their binding in the PTS cascade [ 11 , 12 , 26 , 27 ]. We have also characterized the interaction of EI sc with wild-type HPr sc [ 28 ] and that of the phosphorylated species [ 29 ], as well as that of the N-terminal domain of EI sc (EIN sc ) with intact HPr sc [ 10 , 29 ]. We have also shown that the peptide comprising residues Ala9-Gly30 of HPr sc (HPr 9–30 ) is mainly disordered in aqueous solution (with about 7% of helical population) [ 30 ].…”

“…We have also shown that the peptide comprising residues Ala9-Gly30 of HPr sc (HPr 9–30 ) is mainly disordered in aqueous solution (with about 7% of helical population) [ 30 ]. The peptide binds to the intact EI sc [ 28 ] with a K d of 230 µM, and to EIN sc with a K d of 8 µM (both measured by isothermal titration calorimetry (ITC)) [ 31 ]. The latter value was similar to that measured for the binding reaction between intact HPr sc and EIN sc (12 µM [ 31 ]).…”

Survey of the year 2008: applications of isothermal titration calorimetry

“…In 2008, we identified more than 500 articles after searching Web of Science, PubMed, SciDir and OVID databases using ‘isothermal AND titration AND calorimetry’ or ITC or ‘Isothermal Titration Calorimetry’ search terms. Following the format of previous annual surveys these have been classified into the following categories: References cited in the text and review articles 1–82. Protein–protein and protein–peptide interactions 83–222. Protein–small ligand or protein–drug interactions 223–321. Protein/peptide metal interactions 322–355. Protein/peptide nucleic acid interactions 356–387. Protein/peptide lipid interactions …”