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J Biol Chem. 2017 Jun 2;292(22):9294-9304. doi: 10.1074/jbc.M116.773069. Epub 2017 Apr 10.

Photoaffinity labeling with cholesterol analogues precisely maps a cholesterol-binding site in voltage-dependent anion channel-1.

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From the Departments of Anesthesiology.
Biochemistry and Molecular Biophysics.
the Departments of Physiology and.
the Taylor Family Institute for Innovative Psychiatric Research, Washington University in St. Louis, St. Louis, Missouri 63110.
the Institute for Stem Cell Biology and Regenerative Medicine, Nation Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore 560065 Karnataka, India.
Developmental Biology, and.
Psychiatry, and.
Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine at UCLA, Los Angeles, California 90095, and.
From the Departments of Anesthesiology,


Voltage-dependent anion channel-1 (VDAC1) is a highly regulated β-barrel membrane protein that mediates transport of ions and metabolites between the mitochondria and cytosol of the cell. VDAC1 co-purifies with cholesterol and is functionally regulated by cholesterol, among other endogenous lipids. Molecular modeling studies based on NMR observations have suggested five cholesterol-binding sites in VDAC1, but direct experimental evidence for these sites is lacking. Here, to determine the sites of cholesterol binding, we photolabeled purified mouse VDAC1 (mVDAC1) with photoactivatable cholesterol analogues and analyzed the photolabeled sites with both top-down mass spectrometry (MS), and bottom-up MS paired with a clickable, stable isotope-labeled tag, FLI-tag. Using cholesterol analogues with a diazirine in either the 7 position of the steroid ring (LKM38) or the aliphatic tail (KK174), we mapped a binding pocket in mVDAC1 localized to Thr83 and Glu73, respectively. When Glu73 was mutated to a glutamine, KK174 no longer photolabeled this residue, but instead labeled the nearby Tyr62 within this same binding pocket. The combination of analytical strategies employed in this work permits detailed molecular mapping of a cholesterol-binding site in a protein, including an orientation of the sterol within the site. Our work raises the interesting possibility that cholesterol-mediated regulation of VDAC1 may be facilitated through a specific binding site at the functionally important Glu73 residue.


cholesterol; lipid binding protein; photoaffinity labeling; protein drug interaction; voltage-dependent anion channel (VDAC)

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