Introduction
Leucine and isoleucine are two amino acids that are identical except for
the position of one methyl group, which is attached at the γ-carbon in
Leu and the β-carbon in Ile. The high similarity raises the question to
which degree these two amino acids are used differently in proteins by
nature. Most well-known is the fact that Ile has a lower propensity to
be within α-helices, due to steric clashes caused by the
β-branching.1,2 However, little is known if there are
additional general trends that distinguish the two amino acids within
proteins. We were interested in whether differences exist between these
two amino acids within the structures and sequences of class A G
protein-coupled receptors (GPCRs).
GPCRs are eukaryotic membrane proteins that possess a transmembrane
domain (TMD) consisting of seven plasma membrane spanning α-helices
(TM1-7). These proteins are receptors that detect a variety of GPCR
subtype-specific extracellular signals, ranging from photons over small
organic molecules to proteins. Absorption of a photon or binding of a
molecule leads to conformational rearrangements that activate the
receptor. Active GPCRs transmit the received signal further to cellular
transducers such as G proteins and β-arrestins, which in turn initiate
specific signaling cascades. GPCRs are commonly divided into different
classes (A to F) based on sequence homology.3,4 Class
A (or rhodopsin-like) GPCRs are the most abundant and diverse receptors
and include the most thoroughly studied GPCRs.5 Since
GPCRs span the hydrophobic environment of the plasma membrane, there is
no partitioning into hydrophobic core and hydrophilic shell as present
in soluble proteins. Rather, TMDs of GPCRs need to maintain favorable
interactions with lipids and between TMs, and they need to enable the
correct insertion into the membrane during protein
translation.6-9 These factors add different restraints
on the primary sequence and lead to a general increase in hydrophobicity
of GPCRs and other membrane proteins in comparison to soluble proteins.
One way to determine the overall hydrophobicity of an entire protein or
a stretch of an amino acid sequence is by applying the Wimley-White
whole-residue hydrophobicity scales.10,11 These scales
are based on the change in free energy (ΔG) for the transfer of amino
acids from water to a bilayer interface (ΔGwif) and from
water to octanol (ΔGwoct). Negative values for either
indicate that an amino acid is hydrophobic in the sense that it
energetically disfavors to be in water. The difference between both
values (ΔGwoct-ΔGwif) captures the
change in free energy for the insertion into a membrane. Negative values
indicate that an amino acid favors the aliphatic environment of octanol
over the membrane interface and thus favors the insertion into a
membrane. For conciseness, we refer to the difference in octanol and
interface scales (ΔGwoct-ΔGwif) as
hydropathy. Amino acid sequence stretches with negative hydropathy
typically indicate transmembrane elements. This is used to predict
membrane-spanning elements within membrane protein sequences based on
hydropathy plots.10
We found differences between Leu and Ile within class A GPCR structures
with respect to packing density and protein-surface exposure of the side
chains. Leu residues are more commonly found at the receptor surface and
in less densely packed areas of the receptor. We explored the idea that
Leu adopts a role in adjusting TMD hydropathy and shows thus differences
in these structural properties compared to Ile. Leu appears in specific
patterns within the amino acid compositions of these GPCR TMDs that
would match this putative role. We further assessed to which extent the
observed patterns could be expected based on a simple numerical model
for amino acid frequencies within TMDs.