Actin and tubulin polypeptide chains acquire their native conformation in the presence of the cytoplasmic chaperonin containing TCP-1 (CCT, also called TRiC) and, in the case of alpha- and beta-tubulin, additional protein cofactors. It has been previously demonstrated that nucleotide exchange and ATP hydrolysis act to switch CCT between conformations that interact either strongly or weakly with unfolded substrates [Melki, R., & Cowan, N.J. (1994) Mol. Cell. Biol. 14, 2895-2904]. The present study further documents the conformational changes and function of CCT. It is first shown, by the use of a range of labeled denatured substrate proteins and a radiolabeled total soluble HeLa cell extract, that CCT in the absence of nucleotides can bind any of a large number of proteins in vitro with high affinity. Second, by the use of denatured labeled beta-actin and beta-tubulin as model substrates for binding to CCT, we demonstrate that the CCT particle can contain two substrate protein chains simultaneously. Third, by electron microscopy, sedimentation velocity, and intrinsic fluorescence measurements, we document the conformational difference between CCT in its ATP- and ADP-bound forms, as well as the change that results from binding of substrate protein. A model summarizes substrate association with CCT and the role of the nucleotide in regulating the affinity of CCT for target proteins.

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Treatment of rabbit sarcoplasmic reticulum Ca 2؉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 Asp 351 and ending in the Lys 605 -Arg 615 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 Ca 2؉ -ATPase as follows: high sensitivity of peptidic bond Arg 505 -Ala 506 to trypsin cleavage, and high reactivity of lysine residue Lys 515 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 Ca 2؉ -ATPase. The fragments also bind Nd 3؉ 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 threedimensional 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.The detailed structure of membrane proteins is only known in a few cases. Among P-type ATPases, which are responsible for active cation transport, the best structure to date is only known to about 14 Å resolution, from cryo-electron microscopy; this is for Ca 2ϩ -ATPase, which catalyzes uptake of Ca 2ϩ from the cytosol to the endoplasmic or sarcoplasmic Ca 2ϩ -storing compartment (1-3). However, large three-dimensional crystals of the protein are not available as yet, and the ATPase structure has been mainly deduced from electron microscopy studies of two-dimensional crystals (3-6), combined with studies by other techniques like chemical labeling, Förster-type resonance energy transfer, immunoreactivity of antibodies with various epitopes, site-directed mutagenesis, and proteolysis studies (reviewed in Refs. 7-9). By comparing structures of Ca 2ϩ -ATPase with and without bound nucleotide, suggestions have been made concerning the likely location of the ATP-binding site in the cytoplasmic portion of this pump (10), but the actual structure of the ATP-binding site and the mechanism of ATP hydrolysis are still largely unknown. Nevertheless, it has been hypothesized that this region of the Ca 2ϩ -ATPase might be organized like water-soluble ...

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