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Defining the Epitope Region of a Peptide from the Streptomyces coelicolor Phosphoenolpyruvate:Sugar Phosphotransferase System Able to Bind to the Enzyme I

Estefanía Hurtado-Gómez ^*, Olga Abián  , F. Javier Muñoz , María José Hernáiz   ^¶, Adrián Velázquez-Campoy  and José L. Neira ^* 

^* Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, 03202 Elche (Alicante), Spain; Instituto de Biocomputación y Física de los Sistemas Complejos, Corona de Aragón, 42, 50009 Zaragoza, Spain; H. C. U. Lozano-Blesa, Instituto Aragonés de Ciencias de la Salud (I+CS-CIBEREHD), Avda. S. Juan Bosco, 15, 50009 Zaragoza, Spain; Grupo de Biotransformaciones, Departamento de Química Orgánica y Farmacéutica, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain; and ^¶ Servicio de Interacciones Moleculares, Parque Científico de Madrid, 28040 Madrid, Spain

Correspondence: Address reprint requests to Adrián Velázquez-Campoy, Instituto de Biocomputación y Física de los Sistemas Complejos, Corona de Aragón, 42, 50009 Zaragoza, Spain. Tel.: 34-976-562215; Fax: 34-976-562215; E-mail: adrianvc{at}unizar.es.; or José L. Neira, Instituto de Biología Molecular y Celular, Edificio Torregaitán, Universidad Miguel Hernández, Avda. del Ferrocaril s/n, 03202, Elche (Alicante), Spain. Tel.: 34-966658459. Fax: 34-966658758. E-mail: jlneira{at}umh.es.

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 -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.