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.