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

Abstract

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...

A free‐energy landscape for the glucagon‐like peptide 1 receptor GLP1R

“…Next, we report an increase in energy when the free G protein transits from closed to open. Studies show that the transition between the states for the free G protein has a small energy gap . The open state, however, becomes more dominant when the G protein is bound to the active receptor, as seen in our energy landscape and in the cited studies above.…”

“…For the receptor, active and inactive conformations were taken from PDB 5VAI and 5VEX (monomer A), respectively. For the G‐protein: open‐ordered, open‐disordered, and closed states were based on 3SN6, 5JS8, and 1GP2, respectively (see our previous study for more details). All amino‐acid sequences were modified to match the sequences from AAR05444.1 ( Homo sapiens GLP1R; 29‐421) for the receptor; EAX11358.1 ( H. sapiens glucagon; 181‐210) for GLP‐1; and NP_000507.1 ( H. sapiens G(s)α; 9‐394), NP_032168.1 ( Mus musculus G(s)β; 2‐340), and NP_034445.1 ( M. musculus G(s)γ; 5‐62) for G protein subunits α, β, and γ; respectively; all accessions are from NCBI GenBank.…”

Ric-8A, a G protein chaperone with nucleotide exchange activity induces long-range secondary structure changes in Gα

“…Notably, Switch III and α3 are consensus effector binding sites of GTP-activated Gα (Sprang et al, 2007) and protection from HDX could reflect a structural stiffening of these elements in the GTP-bound state. The pattern of changes in HDX protection upon transition between GDP and GTPγS-bound states roughly parallel the changes in amide chemical shifts observed in the HSQC spectra of Gαi1 (Goricanec et al, 2016). …”

“…Heteronuclear 1 H- 15 N HSQC NMR spectra of both nucleotide-free transducin α (Gαt) (Abdulaev et al, 2006) and Gαi1 (Goricanec et al, 2016) show these α subunits to be only slightly more dynamic than their GDP-bound counterparts, the former bound to Gβγ, as assessed by the broadening of amide resonances. In contrast, as described above, NMR spectra of rhodopsin:Gαt (Abdulaev et al, 2006) and Gαi1:Ric-8A (Thomas et al, 2011) show considerable line broadening indicative of dynamics in the millisecond-microsecond range.…”

Cell‐permeable high‐affinity tracers for Gq proteins provide structural insights, reveal distinct binding kinetics and identify small molecule inhibitors

“…These receptors utilize Gαβγ proteins for activating or inhibiting intracellular signalling pathways. The mechanisms by which G protein activation occurs are not well understood and currently under intensive investigation by many laboratories (Dror et al, 2015;Goricanec et al, 2016;Hilger, Masureel, & Kobilka, 2018;Smith, Lefkowitz, & Rajagopal, 2018;Venkatakrishnan et al, 2016).…”