Calcium Activation of the Ca-ATPase Enhances Conformational Heterogeneity between Nucleotide Binding and Phosphorylation Domains

Abstract

High-resolution crystal structures obtained in two conformations of the Ca-ATPase suggest that a large-scale rigid-body domain reorientation of approximately 50 degrees involving the nucleotide-binding (N) domain is required to permit the transfer of the gamma-phosphoryl group of ATP to Asp(351) in the phosphorylation (P) domain during coupled calcium transport. However, variability observed in the orientations of the N domain relative to the P domain in the different crystal structures of the Ca-ATPase following calcium activation and the structures of other P-type ATPases suggests the presence of conformational heterogeneity in solution, which may be modulated by contact interactions within the crystal. Therefore, to address the extent of conformational heterogeneity between these domains in solution, we have used fluorescence resonance energy transfer to measure the spatial separation and conformational heterogeneity between donor (i.e., 5-[[2-[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid) and acceptor (i.e., fluorescein 5-isothiocyanate) chromophores covalently bound to the P and N domains, respectively, within the Ca-ATPase stabilized in different enzymatic states associated with the transport cycle. In comparison to the unliganded enzyme, the spatial separation and conformational heterogeneity between these domains are unaffected by enzyme phosphorylation. However, calcium activation results in a 3.4 A increase in the average spatial separation, from 29.4 to 32.8 A, in good agreement with the 4.3 A increase in the distance estimated from high-resolution structures where these sites are respectively separated by 31.6 A (1IWO.pdb) and 35.9 A (1EUL.pdb). Thus, the crystal structures accurately reflect the average solution structures of the Ca-ATPase. These results suggest that the approximation of cytoplasmic domains accompanying calcium transport, as observed from crystal structures, occurs in solution within the context of large amplitude domain motions important for catalysis. We suggest that these domain motions enhance the rates of substrate (ATP) access and product (ADP) egress and the probability of a productive juxtaposition of the gamma-phosphoryl moiety of ATP with Asp(351) on the phosphorylation domain to facilitate enzyme phosphorylation and calcium transport.

2-Color Calcium Pump Reveals Closure of the Cytoplasmic Headpiece with Calcium Binding

“…This change was somewhat smaller in magnitude than the 30+ Å change predicted by the first X-ray crystallographic structures ( Fig. 1 A), but larger than that measured by other FRET studies [8], [9], [29], Most notably, the distance change observed here was opposite in direction compared to early crystal structure predictions [4]. The apparent decrease in FRET distance with Ca also contrasts with previous FRET studies that used reactive dyes as donors/acceptors.…”

Concerted but Noncooperative Activation of Nucleotide and Actuator Domains of the Ca-ATPase upon Calcium Binding

“…Solution structures using probes bound to the N- and P-domains have been measured, and are consistent with high-resolution x-ray structures (9, 10). Coordinated motions of the actuator (A) domain are proposed to facilitate the association between the N- and P-domains necessary for phosphoenzyme formation and calcium occlusion (5).…”

Coil-to-Helix Transition within Phospholamban Underlies Release of Ca- ATPase Inhibition in Response to β-Adrenergic Signaling

“…In this model, PLB constrains the structure of the Ca-ATPase in its unphosphorylated state, so that higher calcium concentrations are necessary to overcome the kinetic barrier associated with the cooperative binding of the second calcium ion and the large scale structural rearrangement the results in an increase in the spatial separation between domain elements of the Ca-ATPase. Further, the calciumdependent reorientation of both the transmembrane helices of the Ca-ATPase and individual cytosolic domains is consistent with observed reductions in site-specific chemical cross-linking between engineered sites on the Ca-ATPase and PLB following calcium activation [45,49,54,68].…”

“…Consistent with FTIR measurements [32], there are no large changes in the secondary structure of PLB associated with binding the CaATPase. Thus to bring sites on the Ca-ATPase known to interact with sites on PLB, it is apparent that PLB stabilizes a pre-existing conformation of Ca-ATPase, whose domains have previously been shown to undergo large amplitude motions that are critical for function [57,58,[67][68][73][74][75][76][77][78][79]. Thus, rather than extending the PLB structure to fit the crystal structure of the ATPase, a reorientation of the nucleotide binding domain of the Ca-ATPase by approximately ten degrees permits an efficient juxtaposition of the PLB structure near sites on the Ca-ATPase previously identified through covalent cross-linking methods to be proximal to one another (Fig.…”