Structure of the complex between the cytosolic chaperonin CCT and phosducin-like protein

Abstract

The three-dimensional structure of the complex formed between the cytosolic chaperonin CCT (chaperonin containing TCP-1) and phosducin (Pdc)-like protein (PhLP), a regulator of CCT activity, has been solved by cryoelectron microscopy. Binding of PhLP to CCT occurs through only one of the chaperonin rings, and the protein does not occupy the central folding cavity but rather sits above it through interactions with two regions on opposite sides of the ring. This causes the apical domains of the CCT subunits to close in, thus excluding access to the folding cavity. The atomic model of PhLP generated from several atomic structures of the homologous Pdc fits very well with the mass of the complex attributable to PhLP and predicts the involvement of several sequences of PhLP in CCT binding. Binding experiments performed with PhLP/Pdc chimeric proteins, taking advantage of the fact that Pdc does not interact with CCT, confirm that both the N- and C-terminal domains of PhLP are involved in CCT binding and that several regions suggested by the docking experiment are indeed critical in the interaction with the cytosolic chaperonin.

Fig. 1.

Two-dimensional average images of negatively stained CCT:PhLP complex. (A) Average image of side views obtained from 243 CCT particles of apo-CCT. (B) Average image obtained from 286 side views of CCT:PhLP complexes. (C) Average image obtained from 4,225 top views of CCT:PhLP complexes. (D and E) Average images of the two types of top views of CCT:PhLP:8g (anti-CCTδ) immunocomplexes (average of 324 and 626 particles). The subunit labeled by the antibody is marked with “δ.” (Scale bar, 100 Å.) A schematic model of the each mode of PhLP binding, with the topology of the CCT subunits according to ref. , accompanies each average image.

Fig. 2.

Three-dimensional reconstruction of the CCT:PhLP complex by cryoelectron microscopy. (A) Top view of the CCT:PhLP complex. (B) Side view of the same volume. (C) Side view of the three-dimensional reconstruction of apo-CCT (23).

Fig. 3.

Docking of the atomic model of PhLP into the three-dimensional reconstruction of the CCT:PhLP complex. (A) Docking of the atomic model of PhLP into the CCT:PhLP volume. (B and C) Two enlarged views of the docking of the PhLP atomic model (drawn in tubes) into the CCT:PhLP complex (depicted in transparent fashion). The red arrow in B indicates a region of the PhLP mass that could be filled by the 50 residues of the N-terminal sequence of PhLP not present in the PhLP atomic model. The green regions in the atomic model of PhLP are those suggested by the docking analysis to be involved in CCT binding.

Fig. 4.

Both domains of PhLP participate in CCT binding. A sequence alignment of rat Pdc, PhLP1, PhLP2, and PhLP3 is shown in A. Conserved residues are indicated with gray boxes, and secondary structural elements for Pdc (12) are indicated above the sequence (H for helix and S for β-strand). Shaded boxes below the structural elements represent regions implicated in CCT binding by the docking analysis. A vertical arrow at residue 154 marks the loop between the N-and C-terminal domains. The PhLP/Pdc(154–301) chimera contains the N-terminal domain of PhLP and the C-terminal domain of Pdc and vice versa for the PhLP/Pdc(1–153) chimera. In B, the binding of these proteins to CCT or Gβγ was determined by immunoprecipitation of the PhLP chimeras and immunoblotting for CCTα and Gβ as described in Materials and Methods. Immunoblots show representative data from three separate experiments. Positions of molecular weight standards are shown on the right.

Fig. 5.

Binding of PhLP/Pdc chimeras within the N-terminal domain to CCT. Chimeras of PhLP within the N-terminal domain were made by inserting Pdc sequence as shown in A. The numbers indicate the residues of PhLP that were replaced with the corresponding Pdc residues and conserved residues within the replacements are located in gray boxes. Binding of these PhLP chimeras to CCT or Gβγ was measured as in Fig. 4. (B) Representative immunoblots for CCTα and Gβ, as well as a graphical representation of the CCTα binding data normalized to wild-type PhLP. Bars represent the mean ± standard error from seven separate experiments. No PhLP was added to the blank sample. The standard lanes contain 700 ng of purified CCT (90 ng of CCTα) or 25 ng of Gβγ (21 ng of Gβ).

Fig. 6.

Binding of PhLP/Pdc chimeras within the C-terminal domain to CCT. Chimeras of PhLP within the C-terminal domain were made by inserting Pdc sequence in the loops between the predicted secondary structural elements as shown in A. The numbers indicate the residues of PhLP that were replaced with the corresponding Pdc residues and conserved residues within the replacements are located in gray boxes. Binding of these PhLP chimeras to CCT or Gβγ was measured as in Fig. 4. (B) Representative immunoblots for CCTα and Gβ, as well as a graphical representation of the CCTα binding data normalized to wild-type PhLP. Bars represent the mean ± standard error from six separate experiments. The lanes contain the same amounts of protein as in Fig. 5.