Characterization of the ATP-Binding Domain of the Sarco(endo)plasmic Reticulum Ca²⁺-ATPase:  Probing Nucleotide Binding by Multidimensional NMR

Abstract

The skeletal muscle sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1a) mediates muscle relaxation by pumping Ca(2+) from the cytosol to the ER/SR lumen. In efforts aimed at understanding the structural basis for the conformational changes accompanying the reaction cycle catalyzed by SERCA1a, we have studied the ATP-binding domain of SERCA1a in both nucleotide-bound and -free forms by NMR. Limited proteolysis analyses guided us to express a 28 kDa stably folded fragment containing the nucleotide-binding domain of SERCA1a spanning residues Thr357-Leu600. ATP binding activity was demonstrated for this fragment by a FITC competition assay. A nearly complete backbone resonance assignment of this 28 kDa ATP-binding fragment, in both the AMP-PNP-bound and -free forms, was obtained by means of heteronuclear multidimensional NMR techniques. NMR titration experiments with AMP-PNP revealed a confined nucleotide-binding site which coincides with a cytoplasmic pocket region identified in the crystal structure of apo-SERCA1a. These results are consistent with previous site-directed mutagenesis studies of SERCA1a.

Mapping Interactions between the Ca2+-ATPase and Its Substrate ATP with Infrared Spectroscopy

“…Instead the nucleotide binding bands in the amide I region are caused by backbone stretches within well defined and stable structures, since they are hardly affected by 1 H 2 O/ 2 H 2 O exchange (33). In line with this finding of conformational changes in well structured regions, NMR spectroscopy has detected changes of backbone conformation in the N-domain upon AMP binding (60).…”

The Role of the Invariant His-1069 in Folding and Function of the Wilson's Disease Protein, the Human Copper-transporting ATPase ATP7B

“…It revealed that the spatial location of the SEH 1069 PL sequence is equivalent to that of the 438 GEAT 441 segment of Ca 2ϩ -ATPase. The 438 GEAT 441 segment is a part of the nucleotide-binding region of Ca 2ϩ -ATPase and was shown to be in direct proximity to bound ATP (20). Furthermore, even in the E2 state His-1069 is only 18Å away from the Asp residues of the GDGVND motif that are known to be close to the ␤␥-phosphates of the ATP-magnesium complex during catalysis (21).…”

“…His-1069 is located in the ATP-binding domain of WNDP, a protein region that is most conserved in all P-type ATPases. The ATP-binding domains of several P-type ATPases have been extensively characterized, and chemical modification, mutagenesis, and NMR studies have yielded a detailed map of residues important for nucleotide binding in Ca 2ϩ -ATPase (20). However, this information has only limited value for understanding the role of His-1069, because this residue lies in the region of the ATPbinding domain (the so called N-subdomain), the sequence of which is unique for the P 1 -type ATPases.…”

Modulatory ATP Binding Affinity in Intermediate States of E2P Dephosphorylation of Sarcoplasmic Reticulum Ca2+-ATPase

“…In such a state, only the N-domain would be expected to contribute to nucleotide binding, as opposed to tight packing of the nucleotide between the N-and P-domains seen in the Ca 2 E1⅐AMPPCP state (50). In accordance with this hypothesis, the ATP affinity of expressed N-domain from Ca 2ϩ -ATPase or Na ϩ ,K ϩ -ATPase is in the millimolar range (52)(53)(54), rather than the typical submicromolar/ micromolar affinity range of the intact enzymes. The wild typelike high affinity of mutant 4Gi-46/47 for TNP-8N 3 -ATP may then suggest that the N-domain generally is the only critical contributor to the binding of the photolabel, thus again illustrating the notion that the photolabel binds in a way rather different from that of ATP, although the binding sites overlap.…”