PROJECT SUMMARY R01GM105993 The goal of this renewal of R01-GM105993 is to determine the structure and dynamic properties of the complex between Ric-8A and the alpha subunit of the heterotrimeric G protein Gi (Gαi1). Heterotrimeric G proteins modulate cell metabolism, secretion, electrical conductivity, gene transcription, cell division and cellular motility, and therefore are essential to eukaryotic life. Misregulation of G proteins is associated with cancer and a range of other diseases of relevance to general medicine. While most processes controlled by heterotrimeric G proteins occur at cell membranes, recent research has shown that G alpha subunits (Gα) also control certain events in the cell cytoplasm. Important among these is asymmetric cell division, which is essential for embryonic development. Ric-8A is critical regulator of Gα in this process. Ric-8A is a Guanine nucleotide Exchange Factor (GEF) that activates Gα by catalyzing the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP) at the active site of Gα. The intermediate in this reaction is the nucleotide-free Gα:Ric-8A complex. Describing the structural changes that occur when Ric-8A binds to Gα·GDP is key to understanding how Ric-8A activates Gα. Ric-8A is also a chaperone that promotes proper folding of Gα in cells. The two aims of this proposal will test the hypothesis that Gαi1 and possibly Ric-8A sample multiple conformational states in the complex Gαi1:Ric-8A and to make use of structural information to ask whether the GEF and chaperone activities are mechanistically inseparable, or are distinct functions that can be decoupled. The mechanism by which Ric-8A is regulated by protein kinase CK2 will also be investigated. Aim 1 is to determine the structure of Ric-8A and its complex with Gαi1 by X-ray crystallography, with the former in both phosphorylated and non-phosphorylated states. Camelid single chain heavy chain variable domains will be used as crystallization adjuvants. Collaborative heteronuclear NMR experiments will be conducted as an independent approach to define the interface between Ric-8A and GαI and to identify residues in transition among conformational states. Assays of GEF and chaperone activities will be conducted to assess the role of residues that are hypothesized from structural studies as mediators of one or both of these activities. Aim 2 will determine the global structure of Ric-8A:Gαi1 using small angle X-ray scattering and cryo-electron microscopy. Modeling of the lower resolution structures using molecular dynamics and Monte Carlo simulations with X-ray structures of Ric-8A and Gαi1 will be used to detect and identify multiple conformational states of the complex.