A pH-dependent molten globule transition is required for activity of the steroidogenic acute regulatory protein, StAR.
The steroidogenic acute regulatory protein (StAR) simulates steroid biosynthesis by increasing the flow of cholesterol from the outer mitochondrial membrane (OMM) to the inner membrane. StAR acts exclusively on the OMM, and only StAR's carboxyl-terminal alpha-helix (C-helix) interacts with membranes. Biophysical studies have shown that StAR becomes a molten globule at acidic pH, but a physiologic role for this structural transition has been controversial. Molecular modeling shows that the C-helix, which forms the floor of the sterol-binding pocket, is stabilized by hydrogen bonding to adjacent loops. Molecular dynamics simulations show that protonation of the C-helix and adjacent loops facilitates opening and closing the sterol-binding pocket. Two disulfide mutants, S100C/S261C (SS) and D106C/A268C (DA), designed to limit the mobility of the C-helix but not disrupt overall conformation, were prepared in bacteria, and their correct folding and positioning of the disulfide bonds was confirmed. The SS mutant lost half, and the DA mutant lost all cholesterol binding capacity and steroidogenic activity with isolated mitochondria in vitro, but full binding and activity was restored to each mutant by disrupting the disulfide bonds with dithiothreitol. These data strongly support the model that StAR activity requires a pH-dependent molten globule transition on the OMM.
Baker
Bo Y
BY
Department of Pediatrics and Metabolic Research Unit, University of California, San Francisco, California 94143, USA.
Yaworsky
Dustin C
DC
Miller
Walter L
WL
D000595:Amino Acid Sequence; D002784:Cholesterol / Q000737:chemistry; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000378:metabolism; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010750:Phosphoproteins / Q000737:chemistry*; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012984:Software; D013997:Time Factors; D014466:Ultraviolet Rays; D000098682:Steroidogenic Acute Regulatory Protein
D004220:Disulfides; D008565:Membrane Proteins; D010750:Phosphoproteins; D000098682:Steroidogenic Acute Regulatory Protein; D002784:Cholesterol