Molten-globule structure and membrane binding of the N-terminal protease-resistant domain (63-193) of the steroidogenic acute regulatory protein (StAR).
The first step in steroidogenesis is the movement of cholesterol from the outer to inner mitochondrial membrane; this movement is facilitated by the steroidogenic acute regulatory protein (StAR). StAR has molten-globule properties at low pH and a protease-resistant N-terminal domain at pH 4 and pH 8 comprising residues 63-193. To explore the mechanism of action of StAR we investigated the structural properties of the bacterially expressed N-terminal domain (63-193 StAR) using CD, limited proteolysis and NMR. Far- and near-UV CD showed that the amount of secondary structure was greater at acidic than at neutral pH, but there was little tertiary structure at any pH. Unlike 63-193 StAR liberated from N-62 StAR by proteolysis, biosynthetic 63-193 StAR was no longer resistant to trypsin or proteinase K at pH 7, or to pepsin at pH 4. Addition of trifluoroethanol and SDS increased secondary structure at pH 7, and dodecylphosphocholine and CHAPS increased secondary structure at pH 2, pH 4 and pH 7. However, none of these conditions induced tertiary structure, as monitored by near-UV CD or NMR. Liposomes of phosphatidylcholine, phosphatidylserine and their mixture increased secondary structure of 63-193 StAR at pH 7, as monitored by far-UV CD, and stable protein-liposome complexes were identified by gel-permeation chromatography. These results provide further evidence that the N-terminal domain of StAR is a molten globule, and provide evidence that this conformation facilitates the interaction of the N-terminal domain of StAR with membranes. We suggest that this interaction is the key to understanding the mechanism of StAR's action.
Song
M
M
Department of Pediatrics, Building MR-IV, Room 209, University of California San Francisco, San Francisco, CA 94143-0978, USA.
Shao
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H
Mujeeb
A
A
James
T L
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Miller
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D015686:Gene Products, env / Q000737:chemistry / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D000098682:Steroidogenic Acute Regulatory Protein
D015686:Gene Products, env; D008081:Liposomes; D008565:Membrane Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; C093834:peptide fragment P828; D000098682:Steroidogenic Acute Regulatory Protein; C429145:steroidogenic acute regulatory protein (63-193)