Structural Evidence for a Sequential Release Mechanism for Activation of Heterotrimeric G Proteins

G protein ␣ subunits cycle between active and inactive conformations to regulate a multitude of intracellular signaling cascades. Important structural transitions occurring during this cycle have been characterized from extensive crystallographic studies. However, the link between observed conformations and the allosteric regulation of binding events at distal sites critical for signaling through G proteins remain unclear. Here we describe molecular dynamics simulations, bioinformatics analysis, and experimental mutagenesis that identifies residues involved in mediating the allosteric coupling of receptor, nucleotide, and helical domain interfaces of G␣ i . Most notably, we predict and characterize novel allosteric decoupling mutants, which display enhanced helical domain opening, increased rates of nucleotide exchange, and constitutive activity in the absence of receptor activation. Collectively, our results provide a framework for explaining how binding events and mutations can alter internal dynamic couplings critical for G protein function.Heterotrimeric G proteins are key mediators of intracellular signaling pathways that control diverse cellular processes ranging from movement and division to differentiation and neuronal activity (1). G proteins consist of three subunits: G␣, G␤, and G␥. Bound with GDP, G␣ forms an inactive complex with its G␤␥ subunit partners. Interaction with activated receptor (GPCR) 3 promotes the exchange of GDP for GTP on G␣ and its separation from G␤␥. Both isolated G␣ and G␤␥ can then bind and activate or inhibit downstream effectors. GTP hydrolysis deactivates G␣, which subsequently reassociates with G␤␥ completing the cycle. This cycle is further regulated by two classes of additional proteins called regulators of G protein signaling. These function as either GTPase-activating proteins (which promote GTP hydrolysis) or GDP dissociation inhibitors (GDIs, which hinder exchange of GDP for GTP) (2). Important conformational transitions occurring at each stage of this regulated cycle have been characterized from extensive crystallographic studies. These include GDP, GTP analogue, G␤␥, GTPase-activating protein, GDI and most recently GPCR bound complex structures of G␣. However, the link between the observed conformations and the atomic level mechanisms involved in coupling receptor association, G protein activation, and effector interaction remain unclear.All G␣ proteins consist of a catalytic GTP binding Ras-like domain (termed RasD) and a heterotrimeric G protein specific helical domain (HD). Recent principal component analysis (PCA) of 53 available G␣ crystallographic structures identified three major conformationally distinct groups ( Fig. 1 and Ref. 3). These groups correspond to structures with bound GTP analogues, GDP, and GDI (red, green, and blue points in Fig. 1a, respectively). The major variation in the accumulated structures is the concerted displacements of three nucleotide-binding site loops termed the switch regions (SI, SII, and SIII), as well as a relatively small...

supporting

confidence: 86%

Dynamic Coupling and Allosteric Networks in the α Subunit of Heterotrimeric G Proteins

Yao¹,

Malik

Griggs

et al. 2016

“…Thus, these segments appear to be crucial for transferring the molecular signal from the activated receptor to the nucleotide-binding pocket. In support, most of these segments have been demonstrated to be important for GPCR-G protein interactions leading to GDP release (6,(77)(78)(79). Because our simulation analyses indicate that IC2 and IC3 play key roles in G i coupling, detailed structural analyses of these regions in contact with G i are described as follows.…”

Section: ) (Blue Dots)mentioning

confidence: 82%

Molecular Basis of Cannabinoid CB1 Receptor Coupling to the G Protein Heterotrimer Gαiβγ

Shim

Ahn

Kendall

2013

Background: The molecular basis of CB1 coupling to its cognate G protein is unknown. Results: Using an approach combining mutagenesis and molecular dynamics simulations, we identified CB1 residues critical for G protein signaling. Conclusion: Tight interactions between CB1 and the C-terminal helix ␣ 5 of G␣ i are crucial for G protein signaling. Significance: This is the first reported molecular description of CB1 receptor coupling at the receptor-G i interface.

“…In the first, binding of the receptor to the C terminus of G␣ is thought to trigger conformational changes that can be transmitted to the nucleotide-binding pocket via outward rotation and translation of the ␣5 helix and distortion of the ␤6-␣5 loop, a key site of interaction with the guanine ring (8, 21, 29 -31). In the second pathway, the receptor-dependent ␣5 rotation and translation destabilizes the hydrophobic interactions between the ␣5 and ␣1 helices and the ␤2-␤3 strands, which weaken both phosphate and purine binding sites of nucleotide (10,11,20,27,28). In the two proposed activation pathways, the extreme C terminus of the ␣5 helix facilitates both receptor-G protein interaction and G protein activation (2,8,9,32,33).…”

Section: Discussionmentioning

confidence: 99%

“…SEPTEMBER 9, 2016 • VOLUME 291 • NUMBER 37 loop to release GDP from the binding pocket. Perturbation of ␤2-␤3, ␣1, and the Mg ϩ2 binding regions is sufficient to trigger GDP release (10,11,28). However, in the heterotrimeric G protein, the G␤␥ subunit interacts with Switch II and the phosphate binding region, reducing the dynamics of this region.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

A Conserved Hydrophobic Core in Gαi1 Regulates G Protein Activation and Release from Activated Receptor

Kaya¹,

Lokits

Gilbert³

et al. 2016

G protein-coupled receptor-mediated heterotrimeric G protein activation is a major mode of signal transduction in the cell. Previously, we and other groups reported that the ␣5 helix of G␣ i1 , especially the hydrophobic interactions in this region, plays a key role during nucleotide release and G protein activation. To further investigate the effect of this hydrophobic core, we disrupted it in G␣ i1 by inserting 4 alanine amino acids into the ␣5 helix between residues Gln 333 and Phe 334 (Ins4A). This extends the length of the ␣5 helix without disturbing the ␤6-␣5 loop interactions. This mutant has high basal nucleotide exchange activity yet no receptor-mediated activation of nucleotide exchange. By using structural approaches, we show that this mutant loses critical hydrophobic interactions, leading to significant rearrangements of side chain residues His 57 , Phe 189 , Phe 191 , and Phe 336 ; it also disturbs the rotation of the ␣5 helix and the -interaction between His 57 and Phe 189 . In addition, the insertion mutant abolishes G protein release from the activated receptor after nucleotide binding. Our biochemical and computational data indicate that the interactions between ␣5, ␣1, and ␤2-␤3 are not only vital for GDP release during G protein activation, but they are also necessary for proper GTP binding (or GDP rebinding). Thus, our studies suggest that this hydrophobic interface is critical for accurate rearrangement of the ␣5 helix for G protein release from the receptor after GTP binding.Heterotrimeric G proteins, composed of ␣, ␤, and ␥ subunits, act as a molecular switches that turn on intracellular signaling cascades in response to the activation of G protein-coupled receptors by extracellular stimuli. Therefore, G proteins have a critical role in many different cellular responses (1-6).The G␣ subunit binds GDP and forms a tight complex with the G␤␥ subunits. Activated G protein-coupled receptors can catalyze the exchange of GDP for GTP, which leads to the dissociation of the receptor-G protein complex into isolated receptor and G␣ and G␤␥ subunits. Both the G␣ and G␤␥ subunits can then stimulate or inhibit downstream effectors. Signal propagation ceases after the G␣ subunit hydrolyzes GTP, returns to the inactive state, and rebinds to the G␤␥ subunit, regenerating the GDP-bound heterotrimeric state.Previous studies showed that the activated receptor directly interacts with the G protein by binding to the C-terminal ␣5 helix of G␣, inducing a rigid body rotation and translation that pull this helix into a hydrophobic pocket on the receptor (7,8). This leads to the rearrangement of the interfaces between helices ␣5, ␣1, and the ␤2-␤3 strands and between ␣5 and the ␤6-␣5 loop (1, 7, 9 -11). Residue Phe 336 in the ␣5 helix is highly conserved in small (12, 13) and large GTPases (14) in both the animal and plant kingdoms (15-18). Our in silico results predicted that Phe 336 is the most energetically important residue both in maintaining the basal state and in promoting the receptor-bound conformation (6...