Journal Article

The structural basis of the dominant negative phenotype of the Gαi1β1Y2 G203A/A326S heterotrimer

Ping LIU Ming-zhu JIA X Edward ZHOU Parker W DE WAAL Bradley M DICKSON Bo LlU Li HOU Yan-ting YIN Ya n-yong KANG Yi SHI Karsten MELCHER H Eric XU Yi JIANG

2016

Overview

Aim： Dominant negative mutant G proteins have provided critical insight into the mechanisms of G protein-coupled receptor （GPCR） signaling, but the mechanisms underlying the dominant negative characteristics are not completely understood. The aim of this study was to determine the structure of the dominant negative Gαi1β1Y2 G203A/A326S complex （Gi-DN） and to reveal the structural basis of the mutation-induced phenotype of Gαi1β1Y2. Methods： The three subunits of the Gi-DN complex were co-expressed with a baculovirus expression system. The Gi-DN heterotrimer was purified, and the structure of its complex with GDP was determined through X-ray crystallography. Results： The Gi-DN heterotrimer structure revealed a dual mechanism underlying the dominant negative characteristics. The mutations weakened the hydrogen bonding network between GDP/GTP and the binding pocket residues, and increased the interactions in the Gα-GβY interface. Concomitantly, the Gi-DN heterotrimer adopted a conformation, in which the C-terminus of Gai and the N-termini of both the Gβ and Gy subunits were more similar to the GPCR-bound state compared with the wild type complex. From these structural observations, two additional mutations （T48F and D272F） were designed that completely abolish the GDP binding of the Gi-DN heterotrimer. Conclusion： Overall, the results suggest that the mutations impede guanine nucleotide binding and Gα-GβY protein dissociation and favor the formation of the G protein/GPCR complex, thus blocking signal propagation. In addition, the structure provides a rationale for the design of other mutations that cause dominant negative effects in the G protein, as exemplified by the T48F and D272F mutations.