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

Wayland W.L. Cheng, Melissa M. Budelier, Yusuke Sugasawa, Lucie Bergdoll, María Queralt-Martín, William Rosencrans, Tatiana K. Rostovtseva, Zi Wei Chen, Jeff Abramson, Kathiresan Krishnan, Douglas F. Covey, Julian P. Whitelegge, Alex S. Evers

Research output: Contribution to journalArticle

6 Scopus citations

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.

Original languageEnglish
Pages (from-to)1269-1279
Number of pages11
JournalBiochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
Volume1864
Issue number10
DOIs
StatePublished - Oct 2019

Keywords

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

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