Structural Characterization of A. Thaliana Heterotrimeric G-Proteins

Abstract

Heterotrimeric G-proteins, consisting of three distinct subunits Galpha, Gbeta, and Ggamma, constitute one of the most important signal transduction systems in eukaryotes. In mammals they function as molecular switches in responses to external stimuli such as light and drugs. The activation mechanism for the mammalian heterotrimer is well known while in plants it is just inferred by analogy with the mammalian complex. Plant heterotrimeric G-proteins have been shown to be involved in multiple phisiological process, like regulation during development of leaf shape, cell proliferation, lateral root formation, stomatal density as well as control of post germination process (1); despite this, the available set of subunits looks limited compared to their mammalian cognates. In the Arabidopsis genome only one gene is present for the alpha and beta subunit while at least 3 genes have been discovered to belong to gamma subunit providing functional selectivity and specificity to the heterotrimer (2). In order to elucidate the activation mechanism in plants and better understand the G-protein-mediated molecular pathways, a structural investigation of the A. thaliana complex has been undertaken . Heterotrimeric G-protein subunits ( GPA1, AGB1, and AGG2) from A. thaliana were cloned and purified. GPA1 was purified from P. pastoris while AGB1 and AGG2 from E. coli. For all the three subunits SAXS data are avalaible while for AGG2 subunit several crystallization trials have been put in place. Coexpression of AGB1-AGG2 dimer is currently in progress in E. coli expression system. Data from X-ray diffraction and small angle X-ray scattering will be collected in order to be able to move to the next step: the heterotrimer full reconstitution.