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.

1. Introduction 2

2. What is the significance of the shared structural properties of disease-associated protein fibrils? 3

2.1 Mechanism of amyloid fibril formation in vitro 6

2.1.1 In vitro fibril formation involves transient population of ordered aggregates of intermediate stability, or protofibrils 6

3. Toxic properties of protofibrils 7

3.1 Protofibrils, rather than fibrils, are likely to be pathogenic 7

3.2 The toxic protofibril may be a mixture of related species 8

3.3 Morphological similarities of protofibrils suggest a common mechanism of toxicity 9

3.4 Are the amyloid diseases a subset of a much larger class of previously unrecognized protofibril diseases? 9

3.5 Fibrils, in the form of aggresomes, may function to sequester toxic protofibrils 9

4. Amyloid pores, a common structural link among protein aggregation neurodegenerative diseases 10

4.1 Mechanistic studies of amyloid fibril formation reveal common features, including pore-like protofibrils 10

4.1.1 Amyloid-β (Aβ) (Alzheimer's disease) 10

4.1.2 α-Synuclein (PD and diffuse Lewy body disease) 12

4.1.3 ABri (familial British dementia) 13

4.1.4 Superoxide dismutase-1 (amyotrophic lateral sclerosis) 13

4.1.5 Prion protein (Creutzfeldt–Jakob disease, bovine spongiform encephalopathy, etc.) 14

4.1.6 Huntingtin (Huntington's disease) 14

4.2 Amyloidogenic proteins that are not linked to disease also from pore-like protofibrils 15

4.3 Amyloid proteins form non-fibrillar aggregates that have properties of protein channels or pores 15

4.3.1 Aβ ‘channels’ 15

4.3.2 α-Synuclein ‘pores’ 16

4.3.3 PrP ‘channels’ 16

4.3.4 Polyglutamine ‘channels’ 17

4.4 Nature uses β-strand-mediated protein oligomerization to construct pore-forming toxins 17

5. Mechanisms of protofibril induced toxicity in protein aggregation diseases 19

5.1 The amyloid pore can explain the age-association and cell-type selectivity of the neurodegenerative diseases 19

5.2 Protofibrils may promote their own accumulation by inhibiting the proteasome 20

6. Testing the amyloid pore hypothesis by attempting to disprove it 21

7. Acknowledgments 22

8. References 22

Protein fibrillization is implicated in the pathogenesis of most, if not all, age-associated neurodegenerative diseases, but the mechanism(s) by which it triggers neuronal death is unknown. Reductionist in vitro studies suggest that the amyloid protofibril may be the toxic species and that it may amplify itself by inhibiting proteasome-dependent protein degradation. Although its pathogenic target has not been identified, the properties of the protofibril suggest that neurons could be killed by unregulated membrane permeabilization, possibly by a type of protofibril referred to here as the ‘amyloid pore’. The purpose of this review is to summarize the existing supportive circumstantial evidence and to stimulate further studies designed to test the validity of this hypothesis.