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Structural analysis of the Gβ1‐CCT complex provides insight into the mechanism of Gβ1 folding
Author(s) -
Plimpton Rebecca,
Lai Chun Wan J.,
Haines Kara,
Stowell Caleb,
Cuellar Jorge,
MartinBenito Jaime,
Valpuesta Jose M.,
Willardson Barry M.
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.855.5
Subject(s) - biophysics , protein subunit , crystallography , chaperone (clinical) , structural biology , cryo electron microscopy , chemistry , folding (dsp implementation) , protein folding , biochemistry , biology , medicine , engineering , pathology , electrical engineering , gene
The cytosolic chaperonin containing TCP‐1 (CCT) carries out the final folding steps for the G protein ß1 (Gß1) subunit, enabling Gß1 to assemble into the G protein heterotrimer and act in downstream signaling. We employed cryo‐electron (cryo‐EM) microscopy to elucidate the CCT‐Gß1 interaction, using CCT‐Gß1 complexes purified from insect cells. Reconstructions of cryo‐EM images reveal Gß1 bound to the apical domains of two adjacent CCT subunits, CCTß and CCT?. Gß1 forms a globular structure and sits perpendicular to the axis of the CCT toroid. Thus, Gß1 appears to be mostly folded and bound to CCT through one or two of its propeller blades. We identified the Gß1 binding sites on CCTß and CCT? through mutagenesis and co‐immunoprecipitation techniques. Two regions of these CCT subunits contribute strongly to Gß1 binding: a hydrophobic loop and a hydrophobic groove between two helices above the loop. CCT binds many of its substrates through hydrophobic interactions, yet molecular‐level understanding of specific binding contacts has remained elusive. We identify specific sequences in this hydrophobic chaperone‐substrate interaction that serve to stabilize one or two Gß1 propeller blades, thereby allowing the remaining blades to reach their native conformation.