Nucleotide Binding in an Engineered Recombinant Ca2+-ATPase N-Domain

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• 作者：Edgar D. Páez-Pérez ; Valentín De La Cruz-Torres ; José G. Sampedro
• 刊名：Biochemistry
• 出版年：2016
• 出版时间：December 13, 2016
• 年：2016
• 卷：55
• 期：49
• 页码：6751-6765
• 全文大小：870K
• ISSN：1520-4995

文摘

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 (ΔF_max ∼ 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 (ΔF_max ∼ 12%) with a dissociation constant (K_d) 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.