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

Abstract

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) modulates the preferential use of sugars in bacteria. The first proteins in the cascade are common to all organisms (EI and HPr). The active site of HPr involves a histidine (His15) located immediately before the beginning of the first α-helix. The regulator of sigma D (Rsd) protein also binds to HPr. The region of HPr comprising residues Gly9-Ala30 (HPr9–30), involving the first α-helix (Ala16-Thr27) and the preceding active site loop, binds to both the N-terminal region of EI and intact Rsd. HPr9–30 is mainly disordered. We attempted to improve the affinity of HPr9–30 to both proteins by mutating its sequence to increase its helicity. We designed peptides that led to a marginally larger population in solution of the helical structure of HPr9–30. Molecular simulations also suggested a modest increment in the helical population of mutants, when compared to the wild-type. The mutants, however, were bound with a less favorable affinity than the wild-type to both the N-terminal of EI (EIN) or Rsd, as tested by isothermal titration calorimetry and fluorescence. Furthermore, mutants showed lower antibacterial properties against Staphylococcus aureus than the wild-type peptide. Therefore, we concluded that in HPr, a compromise between binding to its partners and residual structure at the active site must exist to carry out its function.

The Immune System of Marine Organisms as Source for Drugs against Infectious Diseases

“…For instance, in this way, molecular docking and dynamics simulations targeting catalytic and regulatory allosteric sites for searching potential competitive and noncompetitive inhibitors, respectively, have been carried out in our groups [18,19]. Likewise, there is the alternative possibility of designing peptides equivalent to relevant lineal interacting-protein regions (i.e., not conformationally arranged), with the final goal of competing for the binding sites from the protein counterparts and thus disrupting the particular molecular pathway [20,21]. For both approaches, detailed molecular structure data of the target isolated proteins and their complexes are required, as well as knowledge of the molecular pathways of both the cellular response and the pathogen infection involved in each particular case [18][19][20][21].…”