Abstract
Resistance to inhibitors of cholinesterase 8A (Ric8A) is an essential regulator of G protein α-subunits (Gα), acting as a guanine nucleotide exchange factor and a chaperone. We report two crystal structures of Ric8A, one in the apo form and the other in complex with a tagged C-terminal fragment of Gα. These structures reveal two principal domains of Ric8A: an armadillo-fold core and a flexible C-terminal tail. Additionally, they show that the Gα C-terminus binds to a highly-conserved patch on the concave surface of the Ric8A armadillo-domain, with selectivity determinants residing in the Gα sequence. Biochemical analysis shows that the Ric8A C-terminal tail is critical for its stability and function. A model of the Ric8A/Gα complex derived from crosslinking mass spectrometry and molecular dynamics simulations suggests that the Ric8A C-terminal tail helps organize the GTP-binding site of Gα. This study lays the groundwork for understanding Ric8A function at the molecular level.
Highlights
Resistance to inhibitors of cholinesterase 8A (Ric8A) is an essential regulator of G protein αsubunits (Gα), acting as a guanine nucleotide exchange factor and a chaperone
To identify C-terminal constructs of Gα that are suitable for crystallization with Ric8A, we tested one in which the 11 C-terminal residues of Gαt were fused to the B1 domain of Streptococcal protein G (GB1Gαt340–350) and a second in which the 24 C-terminal residues of Gαt were fused to maltose-binding protein (MBP) (MBP-Gαt327–350)
For Ric8A, we used a construct that is truncated at the C-terminus (Ric8A1–492) and that had been reported to retain the full functionality of Ric8A26
Summary
Resistance to inhibitors of cholinesterase 8A (Ric8A) is an essential regulator of G protein αsubunits (Gα), acting as a guanine nucleotide exchange factor and a chaperone. The fact that the chaperone function of Ric[8] augments G-protein signaling could readily explain many, if not all, biological effects of the protein It remains unclear whether the GEF or chaperone function of Ric[8] proteins is predominant with respect to its biological effects, we reasoned that the structure of the Ric8/Gα complex might hold the key to understanding both. This complex underlies the GEF activity of. Ric[8] and may, be similar to the folding intermediate that is present during the biosynthesis of Gα12
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