Fig. 4: MD-based classification of PrefCoup_Gαi2 and Coup_{Gαi2_βarr1} mutants.

A Location of simulated mutants and their coupling profiles shown on a snake plot (left) and the CB₂R structure (right). PrefCoup_Gαi2 mutants are shown in orange and Coup_{Gαi2_βarr1} in blue (PDB ID: 6KPC). B MD simulations scheme: the initial system was simulated in production conditions for 0.6 µs. After that, 5 replicates were respawned applying random perturbations to the atom speeds. These 5 replicates were evolved for further 0.4 µs reaching a total evolution time of 1 µs. This yields a total simulation time of 2.6 µs per mutant. The respawned replicates (5 × 0.4 µs) are used for data analysis. C Distribution plot of the network size of LigACNs (i.e., number of nodes) for PrefCoup_Gαi2 mutants compared to WT and Coup_{Gαi2_βarr1} mutants. The data for LigACNs including interatomic contacts in the ACN for all 34 mutants and the wild type is found in Supplementary Data 2. The center line in boxplots represents the median while the box boundaries extend from the first (25%) and third (75%) quartile, representing the interquartile range (IQR). Boxplot whiskers extend to 1.5*IQR and outliers are represented as points. Schematic representations illustrate the impact of a mutation on the LigACN_top size. D Principal Component Analysis (PCA) of MD data using contact frequencies for 10 different contact types: 3 types of hydrogen bonds, salt bridges, Pi-cation, Pi-stacking, T-stacking, Van der Waals including hydrophobic interactions, water bridges, extended water bridges. The best-performing PC plane for separating Coup_{Gαi2_βarr1} and PrefCoup_Gαi2 is PC1 versus PC10, selected using a Support Vector Classifier (SVC). The SVC model (blue contour line) encompasses the Coup_{Gαi2_βarr1} mutants in the middle of the plane and three different clusters for the PrefCoup_Gαi2 mutants (orange line) at peripheral regions. The WT control simulations are highlighted in red. Source data are provided as a Source data file.