Leu & Ile in class A GPCRs
We compared Leu and Ile residues based on how densely packed their side chains are and how strongly these side chains are exposed on the protein surface. Since GPCRs are membrane proteins, protein-surface exposure captures contact with the lipid bilayer or solvent, depending on the location of the side chain. Further, side-chain packing density and protein-surface exposure measure overlapping properties of residues within protein structures, i.e. a high level of protein-surface exposure will lead to a small packing density for a given residue. A sample of 216 experimental structures from 95 unique GPCRs indicates that Ile tends to occur in more densely packed regions than Leu (Fig. 1A) and that Leu tends to be more protein-surface exposed than Ile (Fig. 1B). This is true for the majority of receptor structures, with a total of 88.4 % of them displaying more densely packed Ile residues and 81.0 % more protein-surface exposed Leu. This general difference between the two amino acids suggests that Leu and Ile residues tend to occur within different structural contexts within class A GPCRs, with Leu being more prone to interact with the lipids in the membrane bilayer.
Interestingly, significant differences in side-chain packing between active- and inactive-state GPCRs are present for Leu and Ile (Fig. 1C). In both cases, packing density is smaller in active- than in inactive-state structures (Ile: − 8.6 %, Leu: − 4.9 %). This is further accompanied by a less pronounced and non-significant increase in protein-surface exposure of the two amino acids (Ile: + 4.6 %, Leu: + 2.8 %) (SI Fig. S1). GPCR activation leads to conformational changes that allow G proteins to bind. These conformational changes include the outward movement of TM6 and the subsequent opening of a cytosolic crevice that accommodates the C-terminal helix of a G protein.5,19 Active-state GPCR structures are generally solved in presence of G proteins or G protein-alike substitutions20,21, which were not included in the calculation of packing densities. It is therefore likely that the decrease in packing density upon activation reflects the opening-up of the G protein binding pocket. This further matches the more pronounced decrease in packing density for Ile than Leu since this conformational change can be expected to have a stronger impact on buried residues.