A mirror code for protein-cholesterol interactions in the two leaflets of biological membranes

Abstract

Cholesterol controls the activity of a wide range of membrane receptors through specific interactions and identifying cholesterol recognition motifs is therefore critical for understanding signaling receptor function. The membrane-spanning domains of the paradigm neurotransmitter receptor for acetylcholine (AChR) display a series of cholesterol consensus domains (referred to as “CARC”). Here we use a combination of molecular modeling, lipid monolayer/mutational approaches and NMR spectroscopy to study the binding of cholesterol to a synthetic CARC peptide. The CARC-cholesterol interaction is of high affinity, lipid-specific, concentration-dependent, and sensitive to single-point mutations. The CARC motif is generally located in the outer membrane leaflet and its reverse sequence CRAC in the inner one. Their simultaneous presence within the same transmembrane domain obeys a “mirror code” controlling protein-cholesterol interactions in the outer and inner membrane leaflets. Deciphering this code enabled us to elaborate guidelines for the detection of cholesterol-binding motifs in any membrane protein. Several representative examples of neurotransmitter receptors and ABC transporters with the dual CARC/CRAC motifs are presented. The biological significance and potential clinical applications of the mirror code are discussed.

Cholesterol and nicotinic acetylcholine receptor: An intimate nanometer-scale spatial relationship spanning the billion year time-scale

“…This computational finding was challenged by physicochemical lipid monolayer studies using a CARC-carrying peptide and cholesterol. The fact that the F-452/W mutation had no effect led us to suggest that it is the aromatic nature of Phe-452, and not its specific structure, that is required for optimal binding [47], in agreement with previous studies suggesting that CARC motifs could contain any of the three aromatic residues, i.e. Phe, Trp or Tyr [8,49].…”

“…NMR spectroscopy experiments (MAS triple resonance magic-angle spinning deuterium NMR using deuterated Ala471) [47] showed that cholesterol addition to phospholipid bilayers containing a synthetic 13C/15N-labeled peptide (Asp464-Val492 in the intact Torpedo γ TM4) caused a reduction in the rotational motion of the peptide within the bilayer, further reinforcing the cholesterol-mediated peptide oligomerization hypothesis. It is tempting to extrapolate the results of the isolated peptide experiments to the possible role of the CARC domains in situ, where they could play a role in cholesterolmediated oligomerization of the receptor.…”

“…One prognostic outcome of our molecular modelling exercises was the unexpected finding that two cholesterol molecules could actually be accommodated on the same TM domain [47,49]. This inference follows from the vectorial nature of the CRAC and CARC motifs, mirror images on the linear sequence ("apolar" Leu/Val → "basic" Lys/Arg for CRAC and "basic" Lys/Arg → "apolar" Leu/Val for CARC, from the N-terminus to the C-terminus sequence).…”

“…demonstrated the lipid-specificity (CARC recognized cholesterol but not phosphatidylcholine) and the concentration dependency of the binding (saturation was reached for peptide concentrations <10 μM) [47]. NMR spectroscopy experiments (MAS triple resonance magic-angle spinning deuterium NMR using deuterated Ala471) [47] showed that cholesterol addition to phospholipid bilayers containing a synthetic 13C/15N-labeled peptide (Asp464-Val492 in the intact Torpedo γ TM4) caused a reduction in the rotational motion of the peptide within the bilayer, further reinforcing the cholesterol-mediated peptide oligomerization hypothesis.…”

“…It is tempting to extrapolate the results of the isolated peptide experiments to the possible role of the CARC domains in situ, where they could play a role in cholesterolmediated oligomerization of the receptor. The high affinity, lipid-specific and saturable nature of the converging lipid monolayer and NMR spectroscopy studies, together with the molecular modeling simulations, constitute a much sturdier argument for the direct physical interaction of cholesterol with a CARC cholesterol-recognition motif [47].…”