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

Abstract

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 AMP-PNP (adenosine 5'-[beta,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. 15N-backbone-relaxation experiments revealed site-specific changes in backbone dynamics that converge on the central beta-sheet domain. Nucleotide binding produced diverse effects on dynamics, with enhanced mobility observed for Ile369, Cys420, Arg467, Asp568, Phe593 and Gly598, whereas rigidifying effects were found for Ser383, Leu419, Thr484 and Thr532. 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 beta-sheet.

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

“…The N-domain was excited at 295 nm, and fluorescence emission spectra were recorded between 300 and 400 nm. The maximal fluorescence intensity was at 338 nm, which was the same as that reported for the wild-type recombinant N-domain . In contrast, the wild-type N-domain obtained upon treatment of SR Ca 2+ -ATPase with proteinase K had a maximal fluorescence intensity at 334 nm .…”

“…A similar approach was used by Moutin et al; however, they expressed in E. coli an SR Ca 2+ -ATPase segment that they named “the large cytoplasmic loop” (LCL), which included the N-domain and a large segment of the P-domain . In contrast, Abu-Abed et al expressed the wild-type Ca 2+ -ATPase N-domain segment of residues Thr357–Leu600 (without mutations) for spectroscopic and nuclear magnetic resonance (NMR) studies. , …”

“…There is ample experimental evidence of the conformational changes in the SR Ca 2+ -ATPase at different steps of the Ca 2+ transport cycle . It is known that phosphorylation and Ca 2+ and ATP binding generate long-range effects in the topology of SR Ca 2+ -ATPase as a whole . The mechanism of translocation of Ca 2+ into the SR lumen involves large conformational rearrangements among the A-domain, the N-domain, and the P-domain, most of them linked to movements in the TM α-helices. , …”

Nucleotide Activation of the Ca-ATPase

“…Prior to the availability of crystal structures of SERCA with bound nucleotide, NMR spectroscopy identified a handful of residues in an N-domain fragment of SERCA that interacts with ATP (75). NMR further identified coupling of nucleotide binding to internal dynamics of the N domain, with six residues showing increased backbone mobility and four residues showing decreased backbone mobility (76). Thus, ATP binding induces changes in SERCA internal dynamics.…”

Structural Insights into the Nucleotide-Binding Domains of the P1B-type ATPases HMA6 and HMA8 from Arabidopsis thaliana

“…An accurate review of the literature shows that nucleotide binding studies of isolated N-domains of P-type ATPases are scarce. A few data have been reported such as on the rat Na + K + -ATPase from rat [36], the rabbit skeletal muscle Ca 2+ -ATPase SERCA1a [37] and the K + -ATPase KdpB from E . coli [38].…”