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[Preprint]. 2024 Feb 1:2024.01.30.578006.
doi: 10.1101/2024.01.30.578006.

Visualizing the impact of disease-associated mutations on G protein-nucleotide interactions

Kara Anazia  1 , Lucien Koenekoop  2 , Guillaume Ferré  1   3 , Enzo Petracco  1   4 , Hugo Gutiérrez-de-Teran  2 , Matthew T Eddy  1
Affiliations

Visualizing the impact of disease-associated mutations on G protein-nucleotide interactions

Kara Anazia et al. bioRxiv. .

Abstract

Activation of G proteins stimulates ubiquitous intracellular signaling cascades essential for life processes. Under normal physiological conditions, nucleotide exchange is initiated upon the formation of complexes between a G protein and G protein-coupled receptor (GPCR), which facilitates exchange of bound GDP for GTP, subsequently dissociating the trimeric G protein into its Gα and Gβγ subunits. However, single point mutations in Gα circumvent nucleotide exchange regulated by GPCR-G protein interactions, leading to either loss-of-function or constitutive gain-of-function. Mutations in several Gα subtypes are closely linked to the development of multiple diseases, including several intractable cancers. We leveraged an integrative spectroscopic and computational approach to investigate the mechanisms by which seven of the most frequently observed clinically-relevant mutations in the α subunit of the stimulatory G protein result in functional changes. Variable temperature circular dichroism (CD) spectroscopy showed a bimodal distribution of thermal melting temperatures across all GαS variants. Modeling from molecular dynamics (MD) simulations established a correlation between observed thermal melting temperatures and structural changes caused by the mutations. Concurrently, saturation-transfer difference NMR (STD-NMR) highlighted variations in the interactions of GαS variants with bound nucleotides. MD simulations indicated that changes in local interactions within the nucleotide-binding pocket did not consistently align with global structural changes. This collective evidence suggests a multifaceted energy landscape, wherein each mutation may introduce distinct perturbations to the nucleotide-binding site and protein-protein interaction sites. Consequently, it underscores the importance of tailoring therapeutic strategies to address the unique challenges posed by individual mutations.

Keywords: G protein; G protein-coupled receptor (GPCR); GTPase; cancer; molecular dynamics (MD); nuclear magnetic resonance (NMR).

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Conflict of interest statement

Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1.
Figure 1.. Crystal structure of GαS with annotated disease-associated mutations.
A, the GTP-bound GαS crystal structure (PDB: 1AZT)1. GTP is shown in orange stick representation and residues selected for investigation shown in blue stick representation. B, expanded view from the dashed box shown in A. GTP, disease-associated residues, and the Switch 1 and Switch 3 structural regions are annotated.
Figure 2.
Figure 2.. Thermal melting temperatures of GαS and GαS variants determined by variable-temperature circular dichroism spectroscopy.
A, representative CD spectrum of GαS bound to GDP (blue curve) and bound to GTPγS (orange curve). B, thermal unfolding of GαS bound to GDP or GTPγS monitored by variable temperature single wavelength CD spectroscopy. Same color scheme as A. C, histogram of melting temperatures determined from fitting the variable temperature CD data of GαS, mini-GαS and GαS variants obtained in complexes with GDP or GTPγS. The dashed horizontal lines are located at the mean Tm values for GαS in complexes with GDP or GTPγS. Error bars were determined from the standard deviation of triplicate measurements.
Figure 3.
Figure 3.. Analysis of hydrogen bond interactions between switch regions extracted from MD simulations.
A, hydrogen-bond interaction profiles for GαS (labeled ‘WT’), GαS[Q227L], GαS[R228C], and GαS[R258A], focused on interactions between Switch I, Switch II, and Switch III regions. Colored boxes indicate the existence of a persistent hydrogen-bond, determined with different thresholds of occurrence along the given MD simulation, proportional to the color intensity for GDP-bound (blue) or GTP-bound (orange) proteins. B, schematic representation of all hydrogen-bond interactions depicted in the H-bond matrices. C, the structure of GTP-bound GαS with the involved residues from the analysis annotated. Residues from Switch I are colored yellow, Switch II colored blue and Switch III colored red.
Figure 4.
Figure 4.. One-dimensional 1H STD-NMR spectra of GαS and GαS variants in complexes with GDP and GppNHp.
A, the chemical structure of GDP and 1D STD-NMR spectra of complexes with GDP. Protons observed in the STD-NMR spectra are annotated ‘a’ to ‘d’. Below the chemical structure are expanded regions from 1D STD-NMR spectra shown in Figure S8 containing the annotated signals. “Reference” is a 1D 1H NMR spectrum of GDP, “protein only” is an STD-NMR control experiment with a sample containing GαS and buffer but no nucleotide, and “nucleotide only” is a STD-NMR control experiment with a sample containing nucleotide and buffer but no protein. B, the chemical structure of GppNHp and 1D STD-NMR spectra of complexes with GppNHp. Other presentation details are the same as in A. Views of the presented NMR data were expanded from the full spectra shown in Figure S7.
Figure 5.
Figure 5.. Normalized STD amplification factors measured for GDP and GppNHp in complex with GαS or GαS variants.
Normalized STD amplification factors (STD-AF) determined for GDP (blue) and GppNHp (orange) in complex with GαS or GαS variants. The specific protons used to quantify the STD-AF values are annotated on the chemical structure of GDP. STD amplification factors were normalized to the ‘a’ proton. Error bars were determined from the signal-to-noise ratios in the corresponding NMR spectrum.
Figure 6.
Figure 6.. STD-NMR amplification factors mapped onto the chemical structures of GDP and GppNHp for complexes with GαS and GαS variants.
For each proton observed in the STD-NMR spectra, the relative STD-AFs are colored as a percentage of the STD-AF normalized with respect to the ‘a’ proton (same as in Figure 3 and 4) indicated with the black circle. The STD-AFs are colored according to their relative intensities compared to those for the ‘a’ proton: relatively weaker interactions representing < 50% (grey), weak-moderate interactions representing 51% to 99% (yellow), moderate interactions representing 101% to 150% (green) and stronger interactions representing > 150% (red). The white circles indicate that no analysis was performed for those protons due to overlap of nucleotide and background residual protein signals.
Figure 7.
Figure 7.. Variation in the structure and dynamics of the nucleotide binding pocket among GαS and GαS variants.
Nucleotide binding pocket of GαS bound to GTP (shown in orange stick representation, PDB:1AZT) with protons seen in STD-NMR interactions annotated ‘a’ through ‘d’. The color palette for the residues interacting with these protons denotes increasing shades of pink which are proportional to the frequency of the residue as being the closest to the given proton, with darker shades of pink indicating higher frequencies as a function of the mutations explored.
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