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Biochim Biophys Acta Mol Cell Biol Lipids. 2019 Jun 5. pii: S1388-1981(19)30096-4. doi: 10.1016/j.bbalip.2019.06.004. [Epub ahead of print]

Multiple neurosteroid and cholesterol binding sites in voltage-dependent anion channel-1 determined by photo-affinity labeling.

Author information

1
Department of Anesthesiology, Washington University in St. Louis, MO 63110, USA.
2
Department of Anesthesiology, Washington University in St. Louis, MO 63110, USA; Department of Biochemistry and Molecular Biophysics, Washington University in St. Louis, MO 63110, USA.
3
Department of Physiology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA.
4
Section on Molecular Transport, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
5
Department of Anesthesiology, Washington University in St. Louis, MO 63110, USA; Taylor Family Institute for Innovative Psychiatric Research, Washington University in St. Louis, MO 63110, USA.
6
Department of Developmental Biology, Washington University in St. Louis, MO 63110, USA.
7
Department of Anesthesiology, Washington University in St. Louis, MO 63110, USA; Department of Developmental Biology, Washington University in St. Louis, MO 63110, USA; Department of Psychiatry, Washington University in St. Louis, MO 63110, USA; Taylor Family Institute for Innovative Psychiatric Research, Washington University in St. Louis, MO 63110, USA.
8
Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA.
9
Department of Anesthesiology, Washington University in St. Louis, MO 63110, USA; Department of Developmental Biology, Washington University in St. Louis, MO 63110, USA; Department of Psychiatry, Washington University in St. Louis, MO 63110, USA; Taylor Family Institute for Innovative Psychiatric Research, Washington University in St. Louis, MO 63110, USA. Electronic address: eversa@wustl.edu.

Abstract

Voltage-dependent anion channel-1 (VDAC1) is a mitochondrial porin that is implicated in cellular metabolism and apoptosis, and modulated by numerous small molecules including lipids. VDAC1 binds sterols, including cholesterol and neurosteroids such as allopregnanolone. Biochemical and computational studies suggest that VDAC1 binds multiple cholesterol molecules, but photolabeling studies have identified only a single cholesterol and neurosteroid binding site at E73. To identify all the binding sites of neurosteroids in VDAC1, we apply photo-affinity labeling using two sterol-based photolabeling reagents with complementary photochemistry: 5α-6-AziP which contains an aliphatic diazirine, and KK200 which contains a trifluoromethyl-phenyldiazirine (TPD) group. 5α-6-AziP and KK200 photolabel multiple residues within an E73 pocket confirming the presence of this site and mapping sterol orientation within this pocket. In addition, KK200 photolabels four other sites consistent with the finding that VDAC1 co-purifies with five cholesterol molecules. Both allopregnanolone and cholesterol competitively prevent photolabeling at E73 and three other sites indicating that these are common sterol binding sites shared by both neurosteroids and cholesterol. Binding at the functionally important residue E73 suggests a possible role for sterols in regulating VDAC1 signaling and interaction with partner proteins.

KEYWORDS:

Cholesterol; Mass spectrometry; Neurosteroid; Photoaffinity labeling; Protein drug interaction; Voltage-dependent anion channel (VDAC)

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