title: Nucleotide Binding in an Engineered Recombinant Ca2+-ATPase N-Domain.
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%) 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%) 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.
authors.affiliation: Instituto de Física, Universidad Autónoma de San Luis Potosí , Manuel Nava 6, Zona Universitaria, CP, 78290 San Luis Potosí, SLP, Mexico.
authors.forename: Edgar D
authors.initials: ED
authors.lastname: Páez-Pérez
authors.affiliation: Instituto de Física, Universidad Autónoma de San Luis Potosí , Manuel Nava 6, Zona Universitaria, CP, 78290 San Luis Potosí, SLP, Mexico.
authors.forename: Valentín
authors.initials: V
authors.lastname: De La Cruz-Torres
authors.affiliation: Instituto de Física, Universidad Autónoma de San Luis Potosí , Manuel Nava 6, Zona Universitaria, CP, 78290 San Luis Potosí, SLP, Mexico.
authors.forename: José G
authors.initials: JG
authors.lastname: Sampedro
mesh_terms: D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D009711:Nucleotides; D011994:Recombinant Proteins; D000252:Calcium-Transporting ATPases