Structures of the Gβ-CCT and PhLP1–Gβ-CCT complexes reveal a mechanism for G-protein β-subunit folding and Gβγ dimer assembly

Abstract

G-protein signaling depends on the ability of the individual subunits of the G-protein heterotrimer to assemble into a functional complex. Formation of the G-protein βγ (Gβγ) dimer is particularly challenging because it is an obligate dimer in which the individual subunits are unstable on their own. Recent studies have revealed an intricate chaperone system that brings Gβ and Gγ together. This system includes cytosolic chaperonin containing TCP-1 (CCT; also called TRiC) and its cochaperone phosducin-like protein 1 (PhLP1). Two key intermediates in the Gβγ assembly process, the Gβ-CCT and the PhLP1-Gβ-CCT complexes, were isolated and analyzed by a hybrid structural approach using cryo-electron microscopy, chemical cross-linking coupled with mass spectrometry, and unnatural amino acid cross-linking. The structures show that Gβ interacts with CCT in a near-native state through interactions of the Gγ-binding region of Gβ with the CCTγ subunit. PhLP1 binding stabilizes the Gβ fold, disrupting interactions with CCT and releasing a PhLP1-Gβ dimer for assembly with Gγ. This view provides unique insight into the interplay between CCT and a cochaperone to orchestrate the folding of a protein substrate.G-protein | chaperonin | phosducin-like protein | electron cryo-microscopy | cross-linking

In vivo aspects of protein folding and quality control

“…6D), which may influence the folding trajectory of the encapsulated substrate. The TRiC-mediated folding of certain substrates, such as heterotrimeric GTP-binding protein (G protein) b subunits, is enhanced by cooperation with the cochaperone phosducin-like protein (105). Although the structure and conformational cycle of TRiC are now well understood, it remains unclear how exactly the special features of the eukaryotic chaperonin contribute to protein folding.…”

Cross-Linking Mass Spectrometry: An Emerging Technology for Interactomics and Structural Biology

“…In addition, the integration of NHS esters and acidic residue-targeting dihydrazides provides complementary maps of protein interactions as well 85,132 . Other combinations such as lysine-reactive reagents with photo-induced unnatural amino acids have also been successful, due to their orthogonal abilities to target solvent-accessible and solvent-inaccessible regions, respectively 145,146 . Novel heterobifunctional cross-linkers combining lysine-targeting NHS chemistry with non-specific photo-activatable diazarenes (sulfo-SDA, sulfo-SBP) have served to further increase the amount of information obtainable by XL-MS, yielding ‘high-density’ datasets with greater potential for de novo modeling 137,147 .…”

The Mechanism and Function of Group II Chaperonins

“…This should not be taken to mean that the group IIs function in isolation in the cell. On the contrary, the group II chaperonins appear to be at the heart of a complex network of co-chaperones [17] [18, 19] [20, 21]. Notable examples include the hexameric prefoldin complex which is often thought to bind to and prevent aggregation of unfolded substrates before handing them off to the chaperonin [22, 23] and the phosducin-like proteins which have been shown to enhance TRiC-mediated folding of several substrates [21, 24].…”

“…Cancer-linked proteins p53 and STAT3 are known to be TRiC substrates [86, 87]. TRiC has been shown to mediate the folding of a number of beta-propeller rich proteins, including telomerase cofactor TCAB1 [88], the cell cycle regulators CDC20, CDH1 as well as G β subunits of signaling complexes containing WD40 domains [20, 21, 26, 89–92]. TRiC also participates in suppressing aggregation and toxicity of Huntingtin in Huntington’s Disease [93–96] and has recently been linked to susceptibility to Alzheimers’ Disease [97].…”