Molecular architecture of Gα _o and the structural basis for RGS16-mediated deactivation

Abstract

Heterotrimeric G proteins relay extracellular cues from heptahelical transmembrane receptors to downstream effector molecules. Composed of an ␣ subunit with intrinsic GTPase activity and a ␤␥ heterodimer, the trimeric complex dissociates upon receptormediated nucleotide exchange on the ␣ subunit, enabling each component to engage downstream effector targets for either activation or inhibition as dictated in a particular pathway. To mitigate excessive effector engagement and concomitant signal transmission, the G␣ subunit's intrinsic activation timer (the rate of GTP hydrolysis) is regulated spatially and temporally by a class of GTPase accelerating proteins (GAPs) known as the regulator of G protein signaling (RGS) family. The array of G protein-coupled receptors, G␣ subunits, RGS proteins and downstream effectors in mammalian systems is vast. Understanding the molecular determinants of specificity is critical for a comprehensive mapping of the G protein system. Here, we present the 2.9 Å crystal structure of the enigmatic, neuronal G protein G␣ o in the GTP hydrolytic transition state, complexed with RGS16. Comparison with the 1.89 Å structure of apo-RGS16, also presented here, reveals plasticity upon G␣ o binding, the determinants for GAP activity, and the structurally unique features of G␣ o that likely distinguish it physiologically from other members of the larger G␣ i family, affording insight to receptor, GAP and effector specificity.any extracellular cues ranging from photons to neurotransmitters are detected with high specificity by G proteincoupled receptors that in turn elicit an intracellular response by promoting GTP exchange on the ␣ subunit of a heterotrimeric G protein. The heterotrimeric G protein, composed of an ␣ subunit exhibiting endogenous GTPase activity and a heterodimeric ␤␥ subunit, dissociates, enabling each component to activate downstream effectors until GTP is hydrolyzed on the ␣ subunit and the heterotrimeric complex reforms. The ␣ subunit's endogenous GTP hydrolysis rate is relatively slow, therefore the cell uses GTPase accelerating proteins (GAPs) to increase the rate to suit the time scale and magnitude needed for a specific physiological response.The regulators of G protein signaling (RGS) proteins are a class of heterotrimeric G protein GAP first identified in Saccharomyces cerevisiae (Sst2) and Caenorhabditis elegans (Egl10) (1, 2). Studies, both biochemical and structural, have shown an overall preference for RGS domains to bind G␣ subunits in their transition state (mimicked by the analog GDP⅐AlF 4 Ϫ ) and to accelerate GTPase activity by stabilizing the transition state of hydrolysis, thereby optimizing the endogenous GTPase activity of the G␣ subunit without directly contributing to the hydrolytic mechanism (3-5). RGS proteins serve to quench the G protein signal temporally and spatially, either independently, or coupled (in cis or in trans) to an effector (6, 7). Thirty-seven RGS proteins have been identified in the human genome, cataloged into eight subfamilies based o...

Structural characterization of recombinant bovine Goαby spectroscopy and homology modeling

“…6(A)). Even though further structural refinement of this loop is required for meaningful predictions, our results strongly enforce the recent report speculating on the specific role of this region in effector coupling [43]. It is proposed that T117, which is located in this loop, is involved in mouse RGS16 recognition.…”

“…The structure of Goα, however, was not determined until recently. The first crystal structure of Goα in the GTP hydrolytic transition state, in complex with RGS16, was obtained by Slep et al [43].…”

Structural diversity in the RGS domain and its interaction with heterotrimeric G protein α-subunits

“…S5A). Similar to the RGS4/G␣ i1 and RGS16/G␣ o complexes (6,22), the aspartate in this position (Asp-172) in our RGS1/G␣ i1 complex (PDB ID 2GTP) functions as a hydrogen bond acceptor with both the main-chain NH of the G␣ i1 switch-I residue Thr-182 and the side chain from Arg-176 of RGS1 ( Fig. 2A).…”

Therapeutic effects of cell-permeant peptides that activate G proteins downstream of growth factors

“…S1), and therefore, these peptides offer a versatile tool to simultaneously modulate multiple pathways downstream of many RTKs (i.e., broad), even in diseases/processes where upstream and downstream events are incompletely understood (i.e., circumvents the limitations of unknown). Third, because GIV binds preferentially to Gi subfamily members but can discriminate within this subfamily by binding to Gαi subunits but not to the close homolog Gαo (∼75% overall similarity to Gαi1/2/3 subunits) (38), TAT-GIV-CT peptides selectively affect the activation of Gαi1/2/3, but not Gαo (i.e., specific). Fourth, these peptides circumvent the limitation that no promising "druggable" pockets have been identified within GIV's C terminus, and that small molecules that can selectively block this platform are yet to be identified.…”