pH-dependent Interactions of the Carboxyl-terminal Helix of Steroidogenic Acute Regulatory Protein with Synthetic Membranes

Steroidogenic acute regulatory (StAR) protein facilitates import of cholesterol into adrenal and gonadal mitochondria where cholesterol is converted to pregnenolone, initiating steroidogenesis. StAR acts exclusively on the outer mitochondrial membrane (OMM) by unknown mechanisms. To identify StAR domains involved in membrane association, we reacted N-62 StAR with small unilamellar vesicles (SUVs) composed of lipids resembling the OMM. Solvent-exposed domains were digested with trypsin, Asp-N, or pepsin at different pH levels, and StAR peptides protected from proteolysis were identified by mass spectrometry. At pH 4 SUVs completely protected residues 259–282; at pH 6.5 this region was partially digested into 254–272, 254–273, and 254–274. Computer-graphic modeling of N-62 StAR indicated these peptides correspond to the C-terminal α4 helix and that residues Leu275, Thr263, and Arg272 in α4 form stabilizing interactions with Gln128, Asp150, and Asp106 in adjacent loops. CD spectroscopy of a 37-mer model of α4 (residues 247–287) indicated a random coil in aqueous buffer, but in 40% methanol the peptide was α-helical and achieved maximal α-helicity at pH 5.0 in the presence of SUVs. Reacting the 37-mer with diethyl pyrocarbamate incorporated into SUVs increased the number of modified residues. Thus the C-terminal α4 helix is critically involved in the membrane association of StAR with OMM lipids. The membrane association and the α-helical structure of the C terminus in the presence of OMM lipids are also pH-dependent. These results further support StAR undergoing a pH-dependent change in its conformation when interacting with the acidic phospholipid head groups of a membrane.