Characterization of a protease-resistant domain of the cytosolic portion of sarcoplasmic reticulum Ca2+-ATPase. Nucleotide- and metal-binding sites.

Treatment of rabbit sarcoplasmic reticulum Ca2+-ATPase with a variety of proteases, including elastase, proteinase K, and endoproteinases Asp-N and Glu-C, results in accumulation of soluble fragments starting close to the ATPase phosphorylation site Asp351 and ending in the Lys605-Arg615 region, well before the conserved sequences generally described as constituting the "hinge" region of this P-type ATPase (residues 670-760). These fragments, designated as p29/30, presumably originate from a relatively compact domain of the cytoplasmic head of the ATPase. They retain two structural characteristics of intact Ca2+-ATPase as follows: high sensitivity of peptidic bond Arg505-Ala506 to trypsin cleavage, and high reactivity of lysine residue Lys515 toward the fluorescent label fluorescein 5'-isothiocyanate. Regarding functional properties, these fragments retain the ability to bind nucleotides, although with reduced affinity compared with intact Ca2+-ATPase. The fragments also bind Nd3+ ions, leaving open the possibility that these fragments could contain the metal-binding site(s) responsible for the inhibitory effect of lanthanide ions on ATPase activity. The p29/30 soluble domain, like similar proteolytic fragments that can be obtained from other P-type ATPases, may be useful for obtaining three-dimensional structural information on the cytosolic portion of these ATPases, with or without bound nucleotides. From our findings we infer that a real hinge region with conformational flexibility is located at the C-terminal boundary of p29/30 (rather than in the conserved region of residues 670-760); we also propose that the ATP-binding cleft is mainly located within the p29/30 domain, with the phosphorylation site strategically located at the N-terminal border of this domain.