Structural basis of function in heterotrimeric G proteins

1. Introduction 2 2. Heterotrimeric G-protein structure 3 2.1. G-protein α subunit 3 2.2. G-protein βγ dimer 8 2.3. Unique role of Gβ5 in complexes with RGS proteins 9 2.4. Heterotrimer structure 10 2.5. Lipid modifications direct membrane association 11 3. Receptor–G protein complex 11 3.1. Low affinity interactions between inactive receptors (R) and G proteins 11 3.2. Receptor activation exposes the high-affinity G-protein binding site 12 3.3. Receptor–G protein interface 14 3.4. Structural determinants of receptor–G protein specificity 15 3.5. Models of the receptor–G protein complex 17 3.6. Sequential interactions may form the receptor–G protein complex 19 4. Molecular basis for G-protein activation 19 4.1. Potential mechanisms of receptor-catalyzed GDP release 20 4.2. GTP-mediated alteration of the receptor–G protein complex 23 5. Activation of downstream effector proteins 24 5.1. Gα interactions with effectors 24 5.2. Gβγ interactions with effectors and regulatory proteins 26 6. G-protein inactivation 28 6.1. Intrinsic GTPase-activity of Gα 28 6.2. GTPase-activating proteins 30 7. Novel regulation of G-protein signaling 31 8. New approaches to study G-protein dynamics 32 8.1. Nuclear magnetic resonance spectroscopy 32 8.2. Site-directed labeling techniques 33 8.3. Mapping allosteric connectivity with computational approaches 34 8.4. Studies of G-protein function in living cells 36 9. Conclusions 37 10. References 38 Heterotrimeric guanine-nucleotide-binding proteins (G proteins) act as molecular switches in signaling pathways by coupling the activation of heptahelical receptors at the cell surface to intracellular responses. In the resting state, the G-protein α subunit (Gα) binds GDP and Gβγ. Receptors activate G proteins by catalyzing GTP for GDP exchange on Gα, leading to a structural change in the Gα(GTP) and Gβγ subunits that allows the activation of a variety of downstream effector proteins. The G protein returns to the resting conformation following GTP hydrolysis and subunit re-association. As the G-protein cycle progresses, the Gα subunit traverses through a series of conformational changes. Crystallographic studies of G proteins in many of these conformations have provided substantial insight into the structures of these proteins, the GTP-induced structural changes in Gα, how these changes may lead to subunit dissociation and allow Gα and Gβγ to activate effector proteins, as well as the mechanism of GTP hydrolysis. However, relatively little is known about the receptor–G protein complex and how this interaction leads to GDP release from Gα. This article reviews the structural determinants of the function of heterotrimeric G proteins in mammalian systems at each point in the G-protein cycle with special emphasis on the mechanism of receptor-mediated G-protein activation. The receptor–G protein complex has proven to be a difficult target for crystallography, and several biophysical and computational approaches are discussed that complement the currently available structural information to improve models of this interaction. Additionally, these approaches enable the study of G-protein dynamics in solution, which is becoming an increasingly appreciated component of all aspects of G-protein signaling.