Energetic analysis of the rhodopsin–G-protein complex links the α5 helix to GDP release

Alexander, Nathan; Preininger, Anita M.; Kaya, Ali I.; Stein, Richard A.; Hamm, Heidi E.; Meiler, Jens

doi:10.1038/nsmb.2705

Cited by 63 publications

(99 citation statements)

References 44 publications

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“…The relative changes in domain orientation of Gαi1 domains shown here are less pronounced than described in a previous crystal structure of a stimulatory G protein in complex with β2-adrenergic receptor (3), which also contained a nanobody bound to the Gα-Gβ interface, but are in better agreement with more recent studies based on molecular modeling and DEER spectroscopy (13)(14)(15). In addition to these earlier studies, we were able to probe the dynamics and the populations of the associated conformational states.…”

Section: Discussionsupporting

confidence: 73%

“…The helical domain is connected to the nucleotidebinding domain through two flexible linkers, and linker 1 (switch I) undergoes conformational changes upon receptor binding (12). The relative orientation of these two subdomains has been investigated previously at lower resolution by double electron-electron resonance (DEER) spectroscopy (13)(14)(15). One of these studies (15) also used extensive molecular dynamics (MD) simulations to monitor conformational changes within the Gα subunit.…”

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confidence: 99%

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Conformational dynamics of a G-protein α subunit is tightly regulated by nucleotide binding

Goricanec

Stehle

Egloff

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Heterotrimeric G proteins play a pivotal role in the signal-transduction pathways initiated by G-protein-coupled receptor (GPCR) activation. Agonist-receptor binding causes GDP-to-GTP exchange and dissociation of the Gα subunit from the heterotrimeric G protein, leading to downstream signaling. Here, we studied the internal mobility of a G-protein α subunit in its apo and nucleotide-bound forms and characterized their dynamical features at multiple time scales using solution NMR, small-angle X-ray scattering, and molecular dynamics simulations. We find that binding of GTP analogs leads to a rigid and closed arrangement of the Gα subdomain, whereas the apo and GDPbound forms are considerably more open and dynamic. Furthermore, we were able to detect two conformational states of the Gα Ras domain in slow exchange whose populations are regulated by binding to nucleotides and a GPCR. One of these conformational states, the open state, binds to the GPCR; the second conformation, the closed state, shows no interaction with the receptor. Binding to the GPCR stabilizes the open state. This study provides an in-depth analysis of the conformational landscape and the switching function of a G-protein α subunit and the influence of a GPCR in that landscape.H eterotrimeric G proteins are localized at the inner leaflet of the plasma membrane where they convey signals from cellsurface receptors to intracellular effectors (1). Heterotrimeric G proteins consist of two functional units, an α subunit (Gα) and a tightly associated βγ complex. The Gα subunit harbors the guanine nucleotide-binding site. In the inactive GDP-bound state, the Gα subunit is associated with the βγ complex. Exchange of GDP for GTP on the Gα subunit, triggered by interaction with the agonist-bound G-protein-coupled receptor (GPCR), results in a conformational change leading to GDP release and ultimately to GTP binding and subunit dissociation. The complexity of the mechanism by which a GPCR activates the Gα subunit based on available crystal structures has been discussed recently (2, 3). Both the Gα subunit and the βγ subunit interact with downstream effectors and regulate their activity. The intrinsic GTP hydrolysis of the Gα subunit returns the protein to the GDP-bound state, thereby increasing its affinity for the Gβγ subunit, and the subunits reassociate (Fig. 1A), ready for interaction with the agonist-bound GPCR. Throughout this cycle, the Gα subunit is engaged in specific interactions with the GPCR and/or the βγ subunit that stabilize the flexible parts of the protein, e.g., its switch regions. Only the GTP-bound form is stable enough to mediate downstream signaling.Crystallographic (4-9), biochemical (10), and biophysical (11-13) studies have elucidated details of the conformational states of the Gα subunit during the GTPase cycle. The Gα subunit has two structural domains, a nucleotide-binding domain (the Ras-like domain) and a helical domain (the α-H domain) that partially occludes the bound nucleotide (Fig. 1A). Because of this steric considerati...

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Section: Discussionsupporting

confidence: 73%

mentioning

confidence: 99%

Conformational dynamics of a G-protein α subunit is tightly regulated by nucleotide binding

Goricanec

Stehle

Egloff

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Our proposed mechanism involves Phe 336 acting as a relay to transmit conformational changes via strands ␤2 and ␤3 and helix ␣1 to the phosphate-binding loop (5,6). These studies are supported by recently published computational studies (11,19,20). Another critical computational paper from Dror et al (21) used molecular dynamic simulations to suggest that the key events in receptor-mediated G protein activation and GDP release are due to the structural rearrangements of the ␤6-␣5 loop.…”

mentioning

confidence: 51%

“…In the structure of Ins4A, the side chain of His 57 (localized on the end of the ␣1 helix) flips from pointing inside to outside of the core, probably due to the lost network of interactions between Phe 336 (␣5), Phe 189 (␤2), and were predicted in our Rho-G i complex model (Fig. 6F, gray) (19). The ␤2AR-G␣ s complex structure (Fig.…”

Section: Structure Of a G␣ C-terminal Insertion Mutantmentioning

confidence: 99%

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

Kaya¹,

Lokits

Gilbert³

et al. 2016

Journal of Biological Chemistry

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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...

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“…The conserved residue Phe 332 in ␣5 was identified to participate in the paths between ␣5 and RasD-HD interface (supplemental Table S2), and substitution of an equivalent position in G␣11 has been reported to be associated with hypocalcemia (44). The importance of Phe 332 on nucleotide exchange has also been demonstrated in both in vitro experiments and computational energetic analysis (13,17,45,46). The ␣5 residue Asp 333 is solvent-exposed and not directly involved in contact with other structural elements outside of ␣5 but is predicted here to be a prominent on-path site (supplemental Table S2).…”

Section: Journal Of Biological Chemistrymentioning

confidence: 87%

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

Yao¹,

Malik

Griggs

et al. 2016

Journal of Biological Chemistry

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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...

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Energetic analysis of the rhodopsin–G-protein complex links the α5 helix to GDP release

Cited by 63 publications

References 44 publications

Conformational dynamics of a G-protein α subunit is tightly regulated by nucleotide binding

Conformational dynamics of a G-protein α subunit is tightly regulated by nucleotide binding

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

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

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