Structural and functional studies of potential cancer targets
J. W. Tang, M. A. Gorman, Albert G. Frauman, Colin M. House, David D.L. Bowtell, Michael W. Parker · Acta Crystallographica Section A Foundations of Crystallography · 2011
In animals, heterotrimeric guanine nucleotide-binding protein (G protein) signaling is initiated by G protein-coupled receptors (GPCRs), which activate G protein subunits.By contrast, the plant Arabidopsis thaliana lacks canonical GPCRs.Its G protein subunit (AtGPA1) is self-activating in the absence of any receptor or guanine nucleotide exchange factor and features both faster binding of GTP and slower GTP hydrolysis when compared to mammalian G{alpha} proteins.Receptor-independent G proteins must have a fundamentally different mechanism for the control of guanine nucleotide exchange, and thus for G protein activation.To decipher that mechanism, we have combined crystallographic, biophysical, molecular dynamics and mutagenesis studies [1].We show that AtGPA1, like animal G proteins, contains a Ras-like domain that is homologous to 'small' G proteins and an {alpha}-helical domain of less defined function.The guanine nucleotide-binding site is located in a cleft between the two domains.In sharp contrast to animal G proteins, however, our structure revealed pronounced disorder in the {alpha}-helical domain.Subsequent molecular dynamics simulations showed strong anticorrelated movements between the helical domain and the Ras domain, indicating frequent dissociation.Within the helical domain, {alpha}A and {alpha}B helices exhibited the greatest structural fluctuations.These pronounced movements correlate with the high intrinsic activity of AtGPA1.Exchanging the {alpha}-helical domains between AtGPA1 and animal G{alpha}i1, which is not self-activating, conferred kinetic and stability features of one G{alpha} to its counterpart.Using such chimeric constructs, we demonstrate that the AtGPA1 helical domain is necessary for self-activation, and that the {alpha}A helix within it is sufficient to confer self-activation to an animal G protein subunit.Our study reveals the structural basis of the mechanism for G protein activation in Arabidopsis based on the intrinsic mobility of the AtGPA1 {alpha}-helical domain.GPCRs are thought to activate G proteins by a distinctly different mechanism that involves the {alpha}5 helix of the Ras domain and the G{beta}{gamma} dimer to accelerate the dissociation of the guanine nucleotide.However, recently, the {alpha}-helical domain of a mammalian G{alpha} was implicated in conferring specificity of the interaction of G{alpha} with an effector of G{beta}{gamma}, the G protein-activated potassium channel [2].Thus the mechanism for nucleotide exchange found in the plant G protein may be partially retained in animal G proteins.