Figure 5. Mutational tolerance elucidates broad structural features and critical residues of the β₂AR.

(A) Residues within the transmembrane domain colored by their tolerance to particular classes of amino acid substitution. Teal residues are intolerant to both hydrophobic and charged amino acids (globally intolerant), and brown residues are tolerant to hydrophobic amino acids but intolerant to charged amino acids (charge intolerant). The charge-sensitive positions’ side chains are enriched pointing into the membrane, while the globally intolerant positions’ side chains face into the core of the protein (see Figure 5—figure supplement 1). (B) The crystal structure of the hydroxybenzyl isoproterenol-activated state of the β₂AR (PDB: 4LDL) with residues from the mutationally intolerant Clusters 1 and 2 highlighted in maroon. (C) 412 β₂AR residues rank ordered by mutational tolerance at the EC₁₀₀ isoproterenol condition. Residues in known structural motifs (colored points) are significantly more sensitive to mutation than other positions on the protein (p<<0.001). Dashed line demarcates the median of the ranking. The top 15 mutationally intolerant residues are listed and colored by motif association. (D-F) Selected vignettes of residues from the mutationally intolerant UMAP clusters and ranking. (D) W286^6x48 of the CWxP motif and the neighboring G315^7x41 are positioned in close proximity. Substitutions at G315^7x41 are likely to cause a steric clash with W286^6x48 (PDB: 4LDL). (E) An inactive-state water-mediated hydrogen bond network (red) associates N51^1x50 and Y326^7x53 (PDB: 2RH1). Disruption of this network may destabilize the receptor. (F) The ligand-bound orthosteric site surface colored by mutational tolerance. Receptor-ligand contacts with the catecholamine head (present in agonist used in assay) are more intolerant to mutation than those in the hydroxybenzyl tail (not present in agonist used in assay) of the isoproterenol analog depicted in this crystal structure (PDB: 4LDL).

Figure 5—figure supplement 1. Mutational profile suggests side chain orientation and environment.

(A) The crystal structure of the hydroxybenzyl isoproterenol-activated state of the β₂AR (PDB: 4LDL) with residues colored by UMAP cluster identity. (B) Distributions of solvent-accessible surface area (SASA) for each cluster at EC₁₀₀. (C) Hydrophobic versus charge sensitivity across all drug conditions. Points are colored by cluster identity. Residues are defined as globally intolerant to substitution if their hydrophobic and charge sensitivity is greater than 0. Similarly, residues are defined to be uniquely charge sensitive if their hydrophobic sensitivity is less than one and their charge sensitivity is greater than 1 (see Materials and methods). (D) The median SASA is significantly higher for positions in the charge-sensitive clusters when compared to the ones in the intolerant clusters across all drug concentrations (all p<0.0005). This suggests that the charge-sensitive cluster residues point toward the lipid, whereas the ones that are intolerant tend to be buried in the core of the protein.

Figure 5—figure supplement 2. Mutational intolerance of functionally related residues.

(A) Relative activation of an integrated CRE luciferase reporter gene for β2AR missense variants mentioned in the manuscript. (B) Functional consequences of mutation for a set of residues near the G-protein coupling region involved in GPCR activation (Venkatakrishnan et al., 2016). (C) Residues that interact with the catecholamine head (orange) of hydroxybenzyl isoproterenol have significantly lower mutational tolerance than those that interact with the hydroxybenzyl functional group on the tail (purple). In our assay, we screened with isoproterenol lacking the hydroxybenzyl tail. These differences are significantly different at EC₅₀ (p=0.028), EC₁₀₀ (p=0.016), and saturating agonist concentration (p=0.008). (D) Functional consequences of mutation at predicted contacts of a cholesterol binding pocket determined in the timolol-bound structure of the β₂AR inactive state (PDB: 3D4S). As predicted, the highly conserved, across species and class A GPCRs, W158^4x50 is the most constrained residue. The shaded bars represent ±1 standard deviation of the mutational tolerance in the tolerant Cluster 6 (green) or the intolerant Clusters 1 and 2 (red). The mean activity of every mutation at a given position (the mutational tolerance) is shown as a blue bar. (E) Effects of mutations at residues in the Gs interface. (F) Three of the four most intolerant β₂AR residues at the G-protein interface (brown) from the β₂AR-Gs complex crystal structure (PDB: 3SN6), V222^5x61, I135^3x54, and Q229^5x68.