Stability and binding of the phosphorylated species of the N-terminal domain of enzyme I and the histidine phosphocarrier protein from the Streptomyces coelicolor phosphoenolpyruvate:sugar phosphotransferase system

• Published in: Archives of biochemistry and biophysics Vol. 526; no. 1; pp. 44 - 53
• Main Authors: Doménech, Rosa, Martínez-Gómez, Ana Isabel, Aguado-Llera, David, Martínez-Rodríguez, Sergio, Clemente-Jiménez, Josefa María, Velázquez-Campoy, Adrián, Neira, José L.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.10.2012
• Subjects: active sites; adenosine triphosphate; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; binding capacity; carbon; Catalytic Domain; Circular dichroism; enthalpy; Enzyme Stability; Fluorescence; Gram-positive bacteria; histidine; Hydrogen Bonding; Isothermal titration calorimetry; Mutagenesis, Site-Directed; Mutation; NMR; Phosphoenolpyruvate Sugar Phosphotransferase System - chemistry; Phosphoenolpyruvate Sugar Phosphotransferase System - genetics; Phosphoenolpyruvate Sugar Phosphotransferase System - metabolism; Phosphoproteins - chemistry; Phosphoproteins - genetics; Phosphoproteins - metabolism; Phosphorylation; Protein folding; Protein stability; protein structure; proteins; serine; Serine - metabolism; Streptomyces coelicolor; Streptomyces coelicolor - enzymology; Thermodynamics; Fluorescence; Protein stability; NMR; Isothermal titration calorimetry; Protein folding; Circular dichroism
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2012.07.004

► The phosphorylated species have a lower stability than the unphosphorylated ones. ► Binding of EIsc and HPrsc involves local conformational changes in both proteins. ► Binding between both proteins is entropically driven. ► The complex with the weakest affinity is that between the phosphorylated species. ► The dimerization of the C terminus of EIsc affects binding of HPrsc. The phosphotransferase system (PTS) is involved in the use of carbon sources in bacteria. It is formed by two general proteins: enzyme I (EI) and the histidine phosphocarrier (HPr), and various sugar-specific permeases. EI is formed by two domains, with the N-terminal domain (EIN) being responsible for the binding to HPr. In low-G+C Gram-positive bacteria, HPr becomes phosphorylated not only by phosphoenolpyruvate (PEP) at the active-site histidine, but also by ATP at a serine. In this work, we have characterized: (i) the stability and binding affinities between the active-site-histidine phosphorylated species of HPr and the EIN from Streptomyces coelicolor; and (ii) the stability and binding affinities of the species involving the phosphorylation at the regulatory serine of HPrsc. Our results show that the phosphorylated active-site species of both proteins are less stable than the unphosphorylated counterparts. Conversely, the Hpr-S47D, which mimics phosphorylation at the regulatory serine, is more stable than wild-type HPrsc due to helical N-capping effects, as suggested by the modeled structure of the protein. Binding among the phosphorylated and unphosphorylated species is always entropically driven, but the affinity and the enthalpy vary widely.