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 alpha4 helix and that residues Leu(275), Thr(263), and Arg(272) in alpha4 form stabilizing interactions with Gln(128), Asp(150), and Asp(106) in adjacent loops. CD spectroscopy of a 37-mer model of alpha4 (residues 247-287) indicated a random coil in aqueous buffer, but in 40% methanol the peptide was alpha-helical and achieved maximal alpha-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 alpha4 helix is critically involved in the membrane association of StAR with OMM lipids. The membrane association and the alpha-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.
Department of Pediatrics and the Metabolic Research Unit, University of California San Francisco, 94143, USA.
Dustin C
DC
Yaworsky
Bo Y
BY
Baker
Himangshu S
HS
Bose
Katrina B
KB
Best
Lauren B
LB
Jensen
John D
JD
Bell
Michael A
MA
Baldwin
Walter L
WL
Miller
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D013058:Mass Spectrometry; D008567:Membranes, Artificial; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D000098682:Steroidogenic Acute Regulatory Protein
D008567:Membranes, Artificial; D010750:Phosphoproteins; D011994:Recombinant Proteins; D000098682:Steroidogenic Acute Regulatory Protein