Publication Details
Calcium Activation of the Ca-ATPase Enhances Conformational Heterogeneity Between Nucleotide Binding and Phosphorylation Domains
Citation
Chen B, TC Squier, and DJ Bigelow. 2004. "Calcium Activation of the Ca-ATPase Enhances Conformational Heterogeneity Between Nucleotide Binding and Phosphorylation Domains ." Biochemistry 43(14):4366-4374.
Journal Article
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˚ involving the nucleotide-binding (N) domain is required to permit the transfer of the γ-phosphoryl group of ATP to Asp351 in the phosphorylation (P) domain during coupled calcium transport. However, variability observed in the orientation of the N-domain relative to the P-domain in both different crystal structures of the Ca-ATPase following calcium activation, and 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 (FRET) 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 is unaffected by enzyme phosphorylation. However, calcium-activation results in a 3.4 Å increase in the average spatial separation, which increases from 29.4 to 32.8 Å, in good agreement with the high-resolution structures where these sites are respectively separated by 31.6 Å (1 IWO.pdb) and 35.9 Å (1EUL.pdb). Thus, the crystal structures accurately reflect the average solution structures of the Ca-ATPase. However, there is substantial conformational heterogeneity for all enzyme states measured, indicating that formation of catalytically important transition states involves a subpopulation of enzyme intermediates. 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. These domain motions permit substrate (ATP) access and product (ADP) egress, and enhance the probability of a productive juxtaposition of the γ-phosphoryl moiety of ATP with Asp351 on the phosphosphorylation domain to facilitate enzyme phosphorylation and calcium transport.
