Hydrophobicity profiles in G protein-coupled receptor transmembrane helical domains

Abstract

The lack of a crystallographically derived structure for all but three G (TP [guanosine triphosphate]-binding) protein-coupled receptor (GPCRs) proteins necessitates the use of computationally derived methods to determine their structures. Computational methodologies allow a mechanistic glimpse into GPCR-ligand interactions at a molecular level to better understand the initial steps leading to a protein's biologic functions, ie, protecting the ligands that activate, deactivate, or inhibit the protein, stabilizing protein structure in the membrane's lipid bilayer, and ensuring that the hydrophilic environment within the GPCR-binding pocket is maintained. Described here is a formalism that quantifies the amphiphilic nature of a helix, by determining the effective hydrophobicity (or hydrophilicity) at specific positions around it. This formalism will enable computational protein modelers to position helices so that the functional aspects of GPCRs are adequately represented in the model. Hydro-Eff®, an online tool, allows users to calculate effective helical hydrophobicities.

Figure 1

Equation 1 was used to determine effective hydrophobicity. The helical wheel for a fictitious sequence (in the inset box) was created using an Internet tool (see http://www-nmr.cabm.rutgers.edu/bioinformatics/Proteomic_tools/Helical_ wheel/). The calculation for μ is shown for the first few residues. The numbers for each residue reflect the hydrophobicities for the residues as taken from Eisenberg's consensus hydrophobicity scales. The angles at which effective hydrophobicities, Θ_θare determined are in red on the inside of the wheel. The effective hydrophobicities in blue are on the outside of the helical wheel. The red curve shows the hydrophilic regions as identified by Hydro-Eff®. The blue curve shows the hydrophobic region for this fictitious helical sequence.

Figure 2

A screen capture of the Web page for the Hydro-Eff®tool. The user can enter one or more sequences in the text box. The text on the web page explains the rationale for Equation 1 and how it can be used to determine effective hydrophobicities. Hydro-Eff can be accessed at http://bioinfo.genetics.uab.edu/hydro-eff.pl. Accessing this program requires an internet browser.

Figure 3

A screen capture of results of effective hydrophobicity at different angles (at 20° intervals) on an idealized helical wheel. The user can access results for effective hydrophobicities determined using seven different hydrophobicity scales. The text on the page also provides some information about the different hydrophobicity scales. Hydro-Eff®results are accessible via an internet browser.

Figure 4

Amino acids and side chains for the residues for the transmembrane helices on which the effective hydrophobicity residues for rhodopsin. The green regions (without side chains) in the helices show the direction of the effective hydrophobicity for that helix. The amino acid residue and side chains, highlighted in red, show charged residues in the helices of the protein (Arg, Lys, Asp, Glu, His). This is to illustrate that although the effective hydrophobicities do not necessarily reside on the most charged residue, equation 1 takes into account the charged residues in its determination of Θ_θ which is pointed in roughly the same direction as the polar side chains. Amino acids in green with side chains showing are those where the effective hydrophobicity Θ_θ resides specifically on a polar amino acid residue. The space-filled blue residue is Lys296 implicated in a covalent bonding with retinal. This residue is pointed into the interior of the protein, where ligand binding is likely to take place.

Figure 5

Amino acids and side chains for residues of transmembrane helices on which are located the effective hydrophobicity residues for the beta adrenergic receptor. The green regions on the helices show the direction of the effective hydrophobicity for that helix. The amino acid residue and side chains, highlighted in red, show charged residues in the helices of the protein (Arg, Lys, Asp, Glu, His). This is to illustrate that although the effective hydrophobicities do not necessarily reside on the most charged residue, equation 1 takes into account the charged residues in its determination of Θ_θ which is pointed in roughly the same direction as the polar side chains. Amino acids in green with side chains showing are those where the effective hydrophobicity Θ_θ resides specifically on a polar amino acid residue. The residues represented by blue space-filled side chains are implicated in ligand binding.

Figure 6

Amino acids and side chains for residues of transmembrane helices on which are located the effective hydrophobicity residues for the adenosine A_2A receptor. The green regions in the helices show the direction of the effective hydrophobicity for that helix. The amino acid residue and side chains, highlighted in red, show charged residues in the helices of the protein (Arg, Lys, Asp, Glu, His). This is to illustrate that although the effective hydrophobicities do not necessarily reside on the most charged residue, equation 1 takes into account the charged residues in its determination of Θ_θ and the effective hydrophobicities are pointed in roughly the same direction as the polar side chains. Amino acids in green with side chains showing are those where the Θ_θ resides specifically on a charged residue. The blue space-filled side chains are for residues that are implicated in ligard binding. The figure shows that these residues are pointed into the interior of the protein where ligand binding is likely to take place.

Figure 7

Structure of rhodopsin with helices truncated to regions that will be embedded in the plasma membrane. The effective hydrophobicities were then determined again using all the scales determined in Hydro-Eff®. The different colors indicate Θ_θ for different hydrophobicity scales, ie, eisenberg consensus scales (red), Von Heinje (blue), Janin (yellow), Chothia (green), Wolfenden (pink), Kyte (orange), and Argos (purple). Despite some outliers, the figures suggest that during computational protein modeling, the helices should be rotated based on hydrophobicities of helices determined on truncated sequences that are likely to be embedded in the plasma membrane. effective hydrophilicities are not necessarily pointed towards the interior of the protein, but might be pointed towards other helices, which might sustain the helical system.