title: The structural basis of the dominant negative phenotype of the Gαi1β1γ2 G203A/A326S heterotrimer.
abstract: OBJECTIVE
  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 Gαi1β1γ2 G203A/A326S complex (Gi-DN) and to reveal the structural basis of the mutation-induced phenotype of Gαi1β1γ2.
  
  
  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-Gβγ interface. Concomitantly, the Gi-DN heterotrimer adopted a conformation, in which the C-terminus of Gαi and the N-termini of both the Gβ and Gγ 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.
  
  
  CONCLUSIONS
  Overall, the results suggest that the mutations impede guanine nucleotide binding and Gα-Gβγ 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.
authors: VARI-SIMM Center, Center for Structure and Function of Drug Targets, CAS-Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors: Ping
authors: P
authors: Liu
authors: VARI-SIMM Center, Center for Structure and Function of Drug Targets, CAS-Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors: Ming-Zhu
authors: MZ
authors: Jia
authors: Laboratory of Structural Sciences, Van Andel Research Institute, Grand Rapids, MI 49503, USA.
authors: X Edward
authors: XE
authors: Zhou
authors: Laboratory of Structural Sciences, Van Andel Research Institute, Grand Rapids, MI 49503, USA.
authors: Parker W
authors: PW
authors: De Waal
authors: Center for Epigenetics, Van Andel Research Institute, Grand Rapids, MI 49503, USA.
authors: Bradley M
authors: BM
authors: Dickson
authors: VARI-SIMM Center, Center for Structure and Function of Drug Targets, CAS-Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors: Bo
authors: B
authors: Liu
authors: VARI-SIMM Center, Center for Structure and Function of Drug Targets, CAS-Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors: Li
authors: L
authors: Hou
authors: VARI-SIMM Center, Center for Structure and Function of Drug Targets, CAS-Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors: Yan-Ting
authors: YT
authors: Yin
authors: Laboratory of Structural Sciences, Van Andel Research Institute, Grand Rapids, MI 49503, USA.
authors: Yan-Yong
authors: YY
authors: Kang
authors: VARI-SIMM Center, Center for Structure and Function of Drug Targets, CAS-Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors: Yi
authors: Y
authors: Shi
authors: Laboratory of Structural Sciences, Van Andel Research Institute, Grand Rapids, MI 49503, USA.
authors: Karsten
authors: K
authors: Melcher
authors: VARI-SIMM Center, Center for Structure and Function of Drug Targets, CAS-Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.|Laboratory of Structural Sciences, Van Andel Research Institute, Grand Rapids, MI 49503, USA.
authors: H Eric
authors: HE
authors: Xu
authors: VARI-SIMM Center, Center for Structure and Function of Drug Targets, CAS-Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors: Yi
authors: Y
authors: Jiang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D002417:Cattle; D018360:Crystallography, X-Ray; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D044387:GTP-Binding Protein beta Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D044388:GTP-Binding Protein gamma Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D056004:Molecular Dynamics Simulation; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D016415:Sequence Alignment; D061987:Sf9 Cells; D018411:Spodoptera
chemical_list: C118981:G-protein Beta gamma; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D043562:Receptors, G-Protein-Coupled; D019206:GTP-Binding Protein alpha Subunits, Gi-Go