A Database of Predicted Binding Sites for Cholesterol on Membrane Proteins, Deep in the Membrane

Abstract

The outer membranes of animal cells contain high concentrations of cholesterol, of which a small proportion is located deep within the hydrophobic core of the membrane. An automated docking procedure is described that allows the characterization of binding sites for these deep cholesterol molecules on the membrane-spanning surfaces of membrane proteins and in protein cavities or pores, driven by hydrogen bond formation. A database of this class of predicted binding site is described, covering 397 high-resolution structures. The database includes sites on the transmembrane surfaces of many G-protein coupled receptors; within the fenestrations of two-pore K⁺ channels and ATP-gated P2X3 channels; in the central cavities of a number of transporters, including Glut1, Glut5, and P-glycoprotein; and in deep clefts in mitochondrial complexes III and IV.

Figure 1

Docking of cholesterol to the agonist-free β₂ adrenergic receptor (PDB: 3D4S). (A) The membrane-spanning surface shows the locations of surface-exposed oxygen (red), nitrogen (blue), and sulfur (yellow) atoms not involved in intramolecular hydrogen bonding. The extracellular (EC) and intracellular (IC) sides of the hydrophobic domain of the membrane surrounding the protein, as given by the OPM database, are shown by red and blue bars, respectively. The central black box shows the position of the 8 Å slab used for docking. The three residues containing surface-exposed, non-hydrogen-bonded O and S atoms located within the box and visible in this view are labeled. (B) The 10 most energetically favorable of the 20 docking poses before selection for hydrogen bonding are shown. The view is from the EC side. (C) The six poses remaining after selection for cholesterol molecules hydrogen bonding to residues not involved in intramolecular hydrogen bonding are shown. These poses involve hydrogen bonding to Ser161 in TM4 and Gly320 in TM7. To see this figure in color, go online.

Figure 2

The two cholesterol-binding sites identified on the agonist-free β₂ adrenergic receptor. (A and B) The TM surface with surface pockets identified using CASTp (20) is shown, colored from most hydrophobic (orange) to most hydrophilic (blue), (A) without and (B) with a cholesterol molecule (green spheres) bound to Gly320. (C) An expanded view of the cholesterol binding pocket (cholesterol in ball and stick representation) is shown. (D) The TM surface with a cholesterol molecule bound to Ser161 is shown. To see this figure in color, go online.

Figure 3

Cholesterol binding within fenestrations of TWIK-1 (PDB: 3UKM). (A) The TM surface showing the pocket around the central fenestration containing a bound cholesterol (green) and the 8 Å search box (black lines) is shown. (B) A tilted view shows the EC plane and the large cavity exposed on the IC side, with the bound cholesterol (green) marked by an arrow. (C) A cutaway view shows a bound cholesterol (green) in the central pore with its −OH group (red) beneath the selectivity filter and its chain in the fenestration. (D) A cutaway view shows a cholesterol molecule with its −OH group in the fenestration. To see this figure in color, go online.

Figure 4

Cholesterol binding within the central pore of the open-state homotrimeric ATP-gated P2X3 channel (PDB: 5SVK). (A) The TM surface showing the large portals connecting the channel pore to the central core of the lipid bilayer is shown with a bound cholesterol (green). (B) A view of the TM domain from the EC side with the EC domains removed shows a cholesterol (spheres) bound in the channel pore. To see this figure in color, go online.

Figure 5

Binding of cholesterol to P-glycoprotein and two mitochondrial complexes. (A) A view of the P-glycoprotein (PDB: 4Q9H) from the IC side shows a bound cholesterol (green), with the two domains colored blue and tan. (B and C) TM surfaces of cytochrome bc1 (PDB: 1BGY) and cytochrome c oxidase (PDB: 1V54), respectively, are given, showing lipid-exposed pockets with bound cholesterol molecules. To see this figure in color, go online.