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.