Discussion
To summarize, Leu side chains generally occur in less densely packed regions and are more protein-surface exposed than Ile side chains in structures of class A GPCRs, indicating that Leu interacts generally more with lipids. Within the TMD sequences of class A GPCRs, Leu decreases the variation in hydropathy between receptors and Leu content correlates with hydropathies calculated without Leu. A simple numerical model was able to reproduce the overall magnitudes of these two patterns when the number of Leu was adjusted to drive the hydropathy toward an optimal value. Taken together, these observations suggest that the hydropathy of class A GPCR TMDs is tuned by Leu. Since hydropathy is a measure for the energetics of membrane insertion, an appropriate Leu content appears to ensure that Class A GPCRs are inserted into membranes and/or are stable within them. The sequence patterns observed with Leu are absent with Ile, indicating that Ile is not involved in adjusting TMD hydropathy.
Leu content and protein hydrophobicity have previously been linked in proteins of thermophiles. In thermophilic organisms, an increased hydrophobicity in the protein core improves thermostability, which keeps these proteins functional at elevated temperatures.22The comparison between 110 pairs of homologous proteins from thermophilic and mesophilic organisms indicated that the Leu content is significantly higher in thermophilic proteins and accounts for a significant change in the aliphatic index.23 The aliphatic index quantifies hydrophobicity based on the Ala, Val, Ile and Leu content of a protein.24 Interestingly, the authors of that study used the correlation between aliphatic index and Leu content to question the validity of the aliphatic index, whereas we would interpret it in a way that the increase in Leu content is the reason for the increased hydrophobicity of these proteins.
One underlying rationale could be that a mutation of any α-helical residue to Leu is less destabilizing than a mutation to the β-branched Ile. Therefore, if an increased protein hydrophobicity is beneficial, then a mutation to Leu might be preserved more commonly than a mutation to Ile, despite their comparable hydrophobicity. In the case of GPCRs, such a stability-driven effect could be further amplified due to the lower intrinsic stability of GPCRs compared to other proteins.25 However, it is unclear to what degree such an effect exists in a membrane environment since α-helix-destabilization by β-branching appears to be absent within membranes, at least for single-span α-helices.26
It is unclear how generalizable the observations made on Class A GPCRs are, particularly because the patterns with Leu are completely absent in GPCRs outside of class A (SI Fig. S2). For these receptors, Leu resembles Ile, whereas Val shows slightly more pronounced correlations that are indicative of hydropathy tuning. Additionally, we considered the entire TMDs as being important for membrane insertion or stability and neglected that residues buried within the TMD are unlikely to contribute to the overall hydropathy. Still, the patterns we observed in the TMD sequences of class A GPCRs remain highly suggestive of Leu tuning the hydropathies of at least this group of proteins. So far, we could not come up with alternative explanations that would produce similar statistical patterns without connecting Leu to hydropathy tuning. To further support the hypothesis that hydropathy is indeed tuned by Leu, and to rule out potential statistical anomalies and alternative explanations, more sophisticated models and alternative approaches need to be explored.