Fig 2: Gß5 folding is initiated by specific contacts between B4 and CCT5.(A) The electrostatic potential map of Gß5 reveals a single surface-exposed basic residue (R269) within B4 (outlined) and surrounding regions. Red, surface-exposed acidic residues; blue, surface-exposed basic residues. (B) The electrostatic potential map of CCT5 reveals a surface-exposed negatively charged pocket that accommodates R269 of Gß5. The interaction is also further stabilized by additional contacts involving D267 and V274. (C) A series of electrostatic, hydrogen-bond and hydrophobic interactions stabilize the position of the outermost ß-hairpin of B4 with CCT5. (D) The R269E mutation increases Gß5 association with CCT with or without RGS9. Note that the R269E charge reversal slows the migration of the variant in SDS gels compared to Gß5 WT causing R269E to co-migrate with a non-specific band in the Gß5 blots. To correct for this, the intensity of the non-specific band in the empty vector lane was subtracted from the R269E band in the quantifications. (E) The R269E mutation reduces Gß5 association with RGS9. Bars represent the average ± standard deviation normalized to the WT Gß5 sample (n = 5). Red *** p < 0.001, **** p < 0.0001 in t-tests compared to WT Gß5.

Fig 3: Reconstructions of Gß5-PhLP1-CCT complexes.(A) Domain organization and structure of folded Gß5. B1–7, Blades 1–7. (B) Views of CCT-PhLP1-Gß5 reconstructions in open, intermediate, and closed states computed from a single dataset. CCT, gray; PhLP1, pink; Gß5, blue. (C) Conformational change in hydrophobic N-terminal arms of CCT5 and CCT7 between open and closed states. Density and model of additional ordered N-terminal residues observed in the closed state of CCT5 and CCT7 are colored red.