Fig. 1. Differences between Leu and Ile in class A GPCRs.(A) Packing density and (B) protein-surface exposure of Leu and Ile side chains in 216 GPCR structures. The diagonals indicate positions where Leu and Ile side chains are equally packed (A) or protein-surface exposed (B). Values above the diagonal indicate that Ile side chains are more densely packed (in A) and that Ile side chains are more protein surface exposed (in B) than Leu side chains. Structures of GPCRs in active and inactive states are highlighted in purple and orange, respectively. (C) Differences in side-chain packing density between inactive- and active-state structures. Error bars indicate 95 % confidence intervals. P-values were determined using Welch’s t-tests and were < 0.001 in both cases. (D)Relative variances of TMD hydropathies. Variances were calculated for hydropathies of complete TMD sequences («All») and for hydropathies of TMD sequences from which the indicated amino acid was removed. Amino acids are ordered from low to high hydropathy. The dashed line indicates the variance of hydropathies of complete sequences as a visual reference. (E) Correlations between amino acid content and TMD hydropathy based on Spearman’s rank correlation coefficient (ρ). Spearman’s ρ ranges from -1 (perfect negative correlation) to +1 (perfect positive correlation) with 0 indicating the absence of any correlation. Negative correlations show that TMD hydropathy decreases (i.e. TMD is more hydrophobic) when the amino acid content increases.(F) Correlations between amino acid content and TMD hydropathy calculated without given amino acid. Positive correlations show that TMD hydropathy (calculated without given amino acid) increases (i.e. TMD is more hydrophilic) when the amino acid content increases. Only correlations for the five amino acids with the lowest hydropathy are shown in E and F.
We are not aware of an obvious reason that accounts for the differences in packing densities and protein-surface exposure between Leu and Ile side chains in class A GPCRs. It is however possible to formulate several hypotheses that could explain the observed differences, and which are not necessarily mutually exclusive. For example, the destabilizing effects due to the β-branching of Ile might require additional structural restraints to be present for adequately accommodating Ile within α-helices, preventing it from being too protein surface exposed. Another underlying rationale could be that Leu might form better interactions with lipids than Ile and thus occurs more often on the protein surface. The hypothesis that intrigued us the most was that Leu is more protein surface exposed because it adjusts the hydropathy of class A GPCRs for optimal insertion into membranes and/or for stability within them.
We hypothesized that whether or not Leu is important for optimizing TMD hydropathy in class A GPCRs could be detected by two patterns with which this amino acid occurs in the overall amino acid composition. The first pattern is based on the spread of values of a property within a population: A property that needs to adopt a defined optimal value will display little variation between different members of the population. If mainly one factor optimizes the value of such a property, then the removal of that factor will lead to a larger variation in the resulting values, since they are no longer optimized. Hence, if Leu is responsible for optimizing TMD hydropathy, then hydropathies calculated without Leu should show larger variations than when calculated including Leu. This was indeed the case for the sequences of 1580 class A GPCR TMDs (Fig. 1D). Among all amino acids, Leu displays the strongest impact on TMD hydropathy variation.
The second expected pattern is related to correlations between Leu content and TMD hydropathy when calculated with and without Leu residues. If Leu tunes hydropathy, then a positive correlation between Leu content and TMD hydropathy (calculated without Leu) is expected because more Leu residues are required to compensate for a more hydrophilic sequence, i.e. the more hydrophilic the TMD of a GPCR is (without Leu), the more Leu residues are required to make this TMD sufficiently hydrophobic (Fig. 1F). This, however, means that the Leu content and the overall TMD hydropathies should be uncorrelated, i.e. the TMD of a GPCR does not become more hydrophobic the more Leu it contains (Fig. 1E). Overall, the correlations between Leu content and TMD hydropathy in the sequence sample match these predictions (Fig. 1E & 1F).
Interestingly, the patterns that are observed with Leu are absent or weaker with Ile, suggesting that Ile is not (or much less) involved in adjusting the hydropathy of the TMDs. This is insofar surprising as both amino acid share similar hydropathies, with Ile (−0.81 kcal/mol) being even slightly more hydrophobic than Leu (−0.69 kcal/mol). However, even if Leu would be the main driving force in adjusting TMD hydropathy in GPCRs, this will not be the only function of Leu. To quantify the extent to which the above-described effects are present when only a part of all Leu residues is involved in hydropathy tuning, we performed numerical simulations based on a simplified model for amino acid compositions.