Homology modeling and biochemical characterization of Gbetagamma dimers

G proteins are heterotrimeric guanine binding proteins that transduce extracellular signals into the cell. It has remained undefined the extent to which the 16 alpha, 5 beta, and 12 gamma subunits combine to form alphabetagamma heterotrimers. The focus of this work was to test the method of homology modeling to make predictions on a subset of heterotrimers, the betagamma dimers. The 60 models were analyzed according to structural characteristics and the interaction interface between beta and gamma or between beta and another protein when betagamma complexed with other proteins were modeled. The 60 models clustered into three sets of dimers: beta5 dimers; dimers with gammas coded to be farnesylated; and the remaining 36 dimers. These clusters suggested differences in behavior of these three sets, but the ability for a particular beta and gamma to dimerize was not discriminated from the modeling information except for the beta5 dimers. The biochemical characteristics of betagamma dimerization were explored in reticulocyte lysate. Two important results came from the biochemical studies: the complete set of 60 dimers which showed for the most part agreement with the literature with a few exceptions; and secondly, that betagamma dimerization is complicated by other interactions taking place between the subunits and proteins within the lysate. One interaction was determined to be between the beta subunits and a chaperone system termed CCT. All 5 betas and the two splice variants bind to the CCT, and this interaction takes place before dimerization. Further work needs to establish the role of the gamma subunit in the CCT-beta interaction and any role for CCT in dimerization. Modeling has the potential to discern differences in betagamma dimers, but the role of another interaction determining which beta-gamma pain become dimers would complicate the ability of homology modeling to make predictions about dimerization. This work lays the groundwork for further development of homology modeling as a tool to understand betagamma dimers with the future possibility of modeling heterotrimers or other interactions with the betagamma dimer. The novel finding of interaction between CCT and beta provides a new avenue to understand G protein heterotrimer formation.