Brigham Young University Provo, Utah, United States
William Ludlam (Brigham Young University)| Joseph Bohman (Brigham Young University)| Ethan Carter (Brigham Young University)| Shuxin Wang (University of Utah)| Sean Zocca (Brigham Young University)| Peter Shen (University of Utah)| Barry Willardson (Brigham Young University)
To perform their physiological functions, nascent G protein β subunits (Gβ) must be folded and assembled into dimers with G protein γ subunits (Gγ) or regulators of G protein signaling (RGS) proteins. They are assisted in this process by the cytosolic chaperonin CCT, which specializes in folding proteins with β-propeller motifs like Gβ. CCT acts in concert with the co-chaperone phosducin-like protein 1 (PhLP1) to complete the Gβ folding and assembly process. To understand this process at the molecular level, we have isolated a folding intermediate of Gβ5 bound to CCT and PhLP1 and determined its structure to 3.2 Å resolution by high-resolution cryo-electron microscopy (cryo-EM). Gβ5 is the least conserved of the five Gβ isoforms, which is recapitulated biochemically and structurally in the way Gβ5 interacts with CCT. Previous studies show that Gβ1 binds to CCT at the rim of the folding chamber and is released from CCT by PhLP1 to interact with Gγ. By contrast, Gβ5 sits deep inside the folding chamber and PhLP1 reaches down from the rim of the folding cavity to stabilize Gβ5 inside the chamber. This structure explains why PhLP1 increases Gβ5 binding to CCT instead of releasing it as is the case with Gβ1. Lastly, we examined CCT-dependent folding of a pathological S81L mutant form of Gβ5 to understand the molecular defect caused by the mutation. Gβ5 S81L showed decreased cellular expression but increased binding to CCT compared to the wildtype, suggesting that the mutation is pathological because it prevents proper folding by CCT. The mutation allowed us to determine a series of structures of the mutant bound to CCT. Over the course of the series, the Gβ5 β-propeller ranges between partially open to closed states, indicating that the mutation likely creates an energy barrier that prevents CCT from bringing the N- and C- terminal portions of the Gβ5 β-propeller together. This finding provides a molecular explanation for the physiological defects in Gβ5 caused by the S81L mutation and represents a breakthrough in visualizing CCT substrate folding dynamics.
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Cryo-electron Microscopy Reconstruction of the Gβ5:PhLP1:CCT folding complex. A) An end on view of the CCT barrel. Gβ5 is visible to one side of the cavity; B) Side view of the complex C) A cutaway of a central slice through the CCT barrel showing a large central mass attributable to Gβ5 and an upper mass at the rim of the cavity attributable to PhLP1. The density have been low pass filtered to 8 Å for better visualization.