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

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.


Table of contents
Page # Figure S1.Purification of GαS protein and functionality.

S-3
Table S1.Summary of biochemical properties of GαS disease-causing variants S-5 Table S2.Primers designed for GαS variants S-6 Figure S2.Thermal melting profile of GαS and diseased variants in GDP and GTPγS determined by circular dichroism.

S-7
Figure S3.Thermal melting profiles of GαS and GαS variants in the presence of GDP, GTPgS, or with no nucleotide added (apo).Table S1.Summary of biochemical properties of GaS disease causing variants.Each variant is characterized in terms of which disease state it is represented in, the location in the GaS structure, the effect of the mutation on the activation of adenylyl cyclase, the rate of GDP dissociation, the rate of GTP binding and the rate of GTPase activity.The dash indicates that this particular value was not determined within the cited study.

Figure S4 .Figure S1 .
Figure S4.Backbone root mean square fluctuations (RMSF) values within the switch regions of GαS and GαS variants in complexes with GDP or GTP S-9

Figure S2 .
Figure S2.Thermal melting profile of GaS and diseased variants in GDP and GTPgS determined by circular dichroism.The thermal unfolding of GaS and diseaseassociated variants bound to GDP and GTPgS monitored by variable temperature single wavelength CD.Same color scheme as Figure 1A.The thermal melting temperatures were obtained by fitting the data to a Boltzmann sigmoidal function in GraphPad prism.

Figure S3 .Figure S4 .
Figure S3.Thermal melting profiles of GaS and GaS variants in the presence of GDP, GTPgS, or with no nucleotide added (apo).A, The thermal unfolding of GaS and variants GaS[R201C], GaS[R258A] and GaS[R265H] when bound to GDP, GTPgS or with no nucleotide added (apo), as monitored by variable temperature single wavelength CD.Same color scheme used as in Figure1.B, histograms of the melting temperature (Tm) values determined by fitting the data shown in panel A. Error bars represent the standard deviation of triplicate measurements.

Figure S5. 1 HFigure S6 .
Figure S5. 1 H signal assignment in reference spectra of GDP and GppNHp.1dimensional 1 H-NMR reference spectrum of GDP (blue) and GppNHp (orange) nucleotide. 1 H signals labeled 'a' through 'd' were utilized in STD-NMR experiments and are shown on the chemical structures of GDP and GppNHp.Assignments were transferred from BMRB entry bmse000270. 1 H signals labeled 1 and 2 are from HEPES buffer, and 1 H signals labeled 3-5 are from the sodium trimethylsilylpropanesulfonate (DSS) NMR standard.8.0 6.0 4.0 2.0 �( 1 H)[ppm] a

Figure S7 .
Figure S7.Optimization of STD-NMR saturation transfer time. 1 H STD-NMR spectra are shown with 40 µM GaS and 2 mM GDP recorded with four different saturation transfer times between 0.5 s and 3.0 s. 1 H signals labeled 'a' through 'd' were used to calculate STD-NMR amplification factors.Expanded views of each signal are shown in the panels at the top.

Figure S8 .
Figure S8.One-dimensional 1 H STD-NMR spectra of GaS and GaS variants in complexes with GDP and GppNHp.STD-NMR spectra of GaS and variants in complex with GDP shown in panel A (in blue) and GppNHp shown in panel B (in orange)."Reference" is a 1D 1 H NMR spectrum of GDP, "protein only" is a STD-NMR control experiment with a sample containing GaS and buffer but no nucleotide, and "nucleotide only" is a STD-NMR control experiment with a sample containing nucleotide and buffer but no protein.Boxed regions indicate areas of interest where an STD-NMR effect is seen.

Figure S9 .
Figure S9.Percent of contact of residues of GaS and GaS variants with GDP and GTP protons in STD-NMR over MD simulations.The percent contact of residues of the GaS protein and GaS[Q227L], GaS[R228C] and GaS[R258A] in contact with the protons "a" to d" (d is the average of chemically equivalent protons) in GDP and GTP over the course of MD simulations.

Table 2 .
Primers designed for GaS variants.List of forward and reverse primers designed to obtain GaS disease-associated variants.