Binding of Steroidogenic Acute Regulatory Protein to Synthetic Membranes Suggests an Active Molten Globule

Abstract

Steroidogenic acute regulatory protein (StAR) mediates cholesterol transport from the outer to the inner mitochondrial membrane during steroid biosynthesis. The mechanism of StAR's action is not established. To address mechanistic issues, we assessed the binding of StAR to artificial membranes by fluorescence resonance energy transfer using endogenous StAR tryptophan residues as the donor and dansyl-phosphatidylethanolamine in the bilayer as the acceptor. Mixing StAR with dansyl-labeled vesicles composed of phosphatidylcholine increased the fluorescence intensity of dansyl emission excited at 280 nm by 10 -40%. This interaction was dependent on pH, with a maximum at pH 3.0 -3.5 and essentially no change above pH 5. Binding experiments at different temperatures and various combinations of phosphatidylcholine, phosphatidylglycerol, cardiolipin, and cholesterol showed that binding involves an electrostatic step and one or more other steps. Although binding prefers a thermodynamically ordered bilayer, the rate-limiting step occurs either when the bilayer is in a fluid state or when there is cholesterol-induced membrane heterogeneity. Experiments with fluorescence and light scattering indicate that StAR binding promotes ordering and aggregation of anionic membranes. The inactive StAR mutant R182L had lower affinity for the membrane, and the partially active mutant L275P had intermediate affinity. Far-UV CD spectroscopy of StAR in PC membranes show more ␤-structure than in aqueous buffers, and the presence of cardiolipin or cholesterol in the membrane fosters a molten globule state. Our data suggest that StAR binds to membranes in a partially unfolded molten globule state that is relevant to the activity of the protein.

A pH-dependent Molten Globule Transition Is Required for Activity of the Steroidogenic Acute Regulatory Protein, StAR

“…We have proposed that this conformational change is induced in vivo by StAR's interaction with protonated phospholipid head groups of the OMM and is required for StAR activity (11,22,24). Consistent with this model, disrupting the mitochondrial proton pump with mCCCP (26) or with lipopolysaccharide (27) abolishes StAR activity, and liposome protection of the C-helix is more complete at pH 4.0 than at pH 6.5 (15).…”

“…Third, a functional mitochondrial proton pump is required for StAR activity; disruption of the mitochondrial electrochemical gradient eliminates StAR-induced steroidogenesis (26,27). Fourth, StAR undergoes a pH-dependent transition to a molten globule in solutions in vitro (24) and in artificial membranes (22,25). Fifth, only limited domains of StAR, encompassing the C-helix and possibly the adjacent ⍀ loops, interact with lipid models of the OMM (15).…”

“…Spectral data and partial proteolysis indicate that StAR undergoes a structural transition to a molten globule at low pH in solution (24) and in association with synthetic membranes (22,25). We have proposed that this conformational change is induced in vivo by StAR's interaction with protonated phospholipid head groups of the OMM and is required for StAR activity (11,22,24).…”

“…First, deletion of 62 amino-terminal residues, including the leader peptide, results in a protein (N-62 StAR) that is confined to the cytoplasm but retains full activity, both in transfected cell systems and in isolated mitochondria in vitro (19 -21). Second, fluorescence resonance energy transfer experiments indicate that StAR directly interacts with synthetic membranes (22). Third, N-62 StAR promotes cholesterol transfer from synthetic vesicles lacking P450scc to those containing P450scc (23).…”

“…Thus, all direct experimental evidence indicates that StAR's site of action is the OMM. Finally, synthetic unilamellar vesicles having a lipid composition that models the OMM only protect StAR's carboxyl-terminal ␣-helix (C-helix) from proteolysis, implying that when StAR interacts with the OMM, most of the protein remains exposed to the cytoplasm (15).Spectral data and partial proteolysis indicate that StAR undergoes a structural transition to a molten globule at low pH in solution (24) and in association with synthetic membranes (22,25). We have proposed that this conformational change is induced in vivo by StAR's interaction with protonated phospholipid head groups of the OMM and is required for StAR activity (11,22,24).…”