Nucleotide Binding in an Engineered Recombinant Ca2 -ATPase N-Domain Article uri icon

abstract

  • A recombinant Ca2 -ATPase nucleotide binding domain (N-domain) harboring the mutations Trp552Leu and Tyr587Trp was expressed and purified. Chemical modification by N-bromosuccinimide and fluorescence quenching by acrylamide showed that the displaced Trp residue was located at the N-domain surface and slightly exposed to solvent. Guanidine hydrochloride-mediated N-domain unfolding showed the low structural stability of the α6-loop−α7 motif (the new Trp location) located near the nucleotide binding site. The binding of nucleotides (free and in complex with Mg2 ) to the engineered N-domain led to significant intrinsic fluorescence quenching (ΔFmax ∼ 30%25) displaying a saturable hyperbolic pattern; the calculated affinities decreased in the following order: ATP > ADP = ADP-Mg2 > ATP-Mg2 . Interestingly, it was found that Ca2 binds to the N-domain as monitored by intrinsic fluorescence quenching (ΔFmax ∼ 12%25) with a dissociation constant (Kd) of 50 μM. Notably, the presence of Ca2 (200 μM) increased the ATP and ADP affinity but favored the binding of ATP over that of ADP. In addition, binding of ATP to the N-domain generated slight changes in secondary structure as evidenced by circular dichroism spectral changes. Molecular docking of ATP to the N-domain provided different binding modes that potentially might be the binding stages prior to γ-phosphate transfer. Finally, the nucleotide binding site was studied by fluorescein isothiocyanate labeling and molecular docking. The N-domain of Ca2 -ATPase performs structural dynamics upon Ca2 and nucleotide binding. It is proposed that the increased affinity of the N-domain for ATP mediated by Ca2 binding may be involved in Ca2 -ATPase activation under normal physiological conditions. © 2016 American Chemical Society
  • A recombinant Ca2%2b-ATPase nucleotide binding domain (N-domain) harboring the mutations Trp552Leu and Tyr587Trp was expressed and purified. Chemical modification by N-bromosuccinimide and fluorescence quenching by acrylamide showed that the displaced Trp residue was located at the N-domain surface and slightly exposed to solvent. Guanidine hydrochloride-mediated N-domain unfolding showed the low structural stability of the α6-loop−α7 motif (the new Trp location) located near the nucleotide binding site. The binding of nucleotides (free and in complex with Mg2%2b) to the engineered N-domain led to significant intrinsic fluorescence quenching (ΔFmax ∼ 30%25) displaying a saturable hyperbolic pattern; the calculated affinities decreased in the following order: ATP > ADP = ADP-Mg2%2b > ATP-Mg2%2b. Interestingly, it was found that Ca2%2b binds to the N-domain as monitored by intrinsic fluorescence quenching (ΔFmax ∼ 12%25) with a dissociation constant (Kd) of 50 μM. Notably, the presence of Ca2%2b (200 μM) increased the ATP and ADP affinity but favored the binding of ATP over that of ADP. In addition, binding of ATP to the N-domain generated slight changes in secondary structure as evidenced by circular dichroism spectral changes. Molecular docking of ATP to the N-domain provided different binding modes that potentially might be the binding stages prior to γ-phosphate transfer. Finally, the nucleotide binding site was studied by fluorescein isothiocyanate labeling and molecular docking. The N-domain of Ca2%2b-ATPase performs structural dynamics upon Ca2%2b and nucleotide binding. It is proposed that the increased affinity of the N-domain for ATP mediated by Ca2%2b binding may be involved in Ca2%2b-ATPase activation under normal physiological conditions. © 2016 American Chemical Society

publication date

  • 2016-01-01