Probing nucleotide-binding effects on backbone dynamics and folding of the nucleotide-binding domain of the sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase

In muscle cells, SERCA (sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase) plays a key role in restoring cytoplasmic Ca2+ levels to resting concentrations after transient surges caused by excitation-coupling cycles. The mechanism by which Ca2+ is translocated to the lumen of the ER (endoplasmic reticulum) involves major conformational rearrangements among the three cytoplasmic domains: actuator (A), nucleotide-binding (N) and phosphorylation (P) domains; and within the transmembrane Ca2+ -binding domain of SERCA. CD, fluorescence spectroscopy and NMR spectroscopy were used in the present study to probe the conformation and stability of the isolated N domain of SERCA (SERCA-N), in the presence and absence of AMPPNP (adenosine 5'- [ss,gamma-imido] triphosphate). CD and tryptophan fluorescence spectroscopy results established that the effects of nucleotide binding were not readily manifested on the global fold and structural stability of SERCA-N. N-15-backbone-relaxation experiments revealed site-specific changes in backbone dynamics that converge on the central P-sheet domain. Nucleotide binding produced diverse effects on dynamics, with enhanced mobility observed for Ile(369), CyS420, Arg(467), Asp(568), Phe(593) and Gly(598), whereas rigidfying effects were found for Ser(383), Leu(419), Thr(484) and Thr(532). These results demonstrate that the overall fold and backbone motional properties of SERCA-N remained essentially the same in the presence of AMP-PNP, yet revealing evidence for internal counter-balancing effects on backbone dynamics upon binding the nucleotide, which propagate through the central ss-sheet.