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.