Two classes of cholesterol binding sites for the β2AR revealed by thermostability and NMR

Deborah L. Gater, Olivier Saurel, Iordan Iordanov, Wei Liu, Vadim Cherezov, Alain Milon

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

Cholesterol binding to G protein-coupled receptors (GPCRs) and modulation of their activities in membranes is a fundamental issue for understanding their function. Despite the identification of cholesterol binding sites in high-resolution x-ray structures of the β2 adrenergic receptor (β2AR) and other GPCRs, the binding affinity of cholesterol for this receptor and exchange rates between the free and bound cholesterol remain unknown. In this study we report the existence of two classes of cholesterol binding sites in β2AR. By analyzing the β2AR unfolding temperature in lipidic cubic phase (LCP) as a function of cholesterol concentration we observed high-affinity cooperative binding of cholesterol with sub-nM affinity constant. In contrast, saturation transfer difference (STD) NMR experiments revealed the existence of a second class of cholesterol binding sites, in fast exchange on the STD NMR timescale. Titration of the STD signal as a function of cholesterol concentration provided a lower limit of 100 mM for their dissociation constant. However, these binding sites are specific for both cholesterol and β2AR, as shown with control experiments using ergosterol and a control membrane protein (KpOmpA). We postulate that this specificity is mediated by the high-affinity bound cholesterol molecules and propose the formation of transient cholesterol clusters around the high-affinity binding sites.

Original languageEnglish (US)
Pages (from-to)2305-2312
Number of pages8
JournalBiophysical Journal
Volume107
Issue number10
DOIs
StatePublished - Nov 18 2014
Externally publishedYes

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Adrenergic Receptors
Binding Sites
Cholesterol
G-Protein-Coupled Receptors
Ergosterol
Membrane Proteins
X-Rays
Temperature
Membranes

ASJC Scopus subject areas

  • Biophysics

Cite this

Two classes of cholesterol binding sites for the β2AR revealed by thermostability and NMR. / Gater, Deborah L.; Saurel, Olivier; Iordanov, Iordan; Liu, Wei; Cherezov, Vadim; Milon, Alain.

In: Biophysical Journal, Vol. 107, No. 10, 18.11.2014, p. 2305-2312.

Research output: Contribution to journalArticle

Gater, Deborah L. ; Saurel, Olivier ; Iordanov, Iordan ; Liu, Wei ; Cherezov, Vadim ; Milon, Alain. / Two classes of cholesterol binding sites for the β2AR revealed by thermostability and NMR. In: Biophysical Journal. 2014 ; Vol. 107, No. 10. pp. 2305-2312.
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