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

Abstract

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 ␣-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.Molten globules are compact, partially unfolded proteins that retain their secondary structure but have lost some tertiary structure (1); such partially unfolded structures are typically inactive but may be intermediates in membrane insertion (2). Steroidogenic acute regulatory protein (StAR), 3 which is essential for normal adrenal and gonadal steroidogenesis, facilitates the flow of cholesterol from the outer mitochondrial membrane (OMM) to the inner mitochondrial membrane (IMM), where cholesterol is converted to pregnenolone by the cholesterol side chain cleavage enzyme, P450scc (3, 4). Mutation of human StAR causes potentially lethal congenital lipoid adrenal hyperplasia (5, 6); all missense mutations that cause this disease are found in the carboxyl-terminal 50% of the protein, indicating that these sequences are required for StAR activity (7-9). StAR is synthesized as a 37-kDa phosphoprotein with an amino-terminal mitochondrial leader sequence that is cleaved during mitochondrial entry (3, 10, 11).The mechanism by which StAR moves cholesterol from the OMM to IMM remains unclear. The x-ray crystal structures of two closely related proteins, N-216 MLN64 (12) and StarD4 (13), reveal a ␤-barrel structure with a hydrophobic sterol-binding pocket (SBP) that will accommodate one cholesterol molecule, although the apparent access channel to the SBP is too small to accommodate a cholesterol molecule. Models of StAR show the same fold (12,14,15), suggesting action as a transport protein. Recent data suggest that members of the StarD4 family of related proteins, which lack mitochondrial targeting sequences, serve as cytosolic transporters for insoluble lipid molecu...

Early steps in steroidogenesis: intracellular cholesterol trafficking

“…The ⍀ -loops form hydrogen bonds with the C-terminal helix; when these bonds are intact, the C-helix obstructs access of cholesterol to StAR's hydrophobic cholesterol-binding pocket. Molecular Dynamics simulations indicated that the C-helix can "swing open," especially when the surface residues are protonated, as would be expected when StAR interacts with the OMM ( 213 ). It is presumed that the interaction of StAR with the charged phospholipid head groups on the OMM disrupts these such as puromycin or cycloheximide, indicating that a short-lived protein species mediates this process (183)(184)(185).…”

“…It was initially noted that the 37 kDa cytoplasmic form possessing the mitochondrial leader sequence had a short half-life of less than 15 min, suggesting it was a "precursor," whereas the 30 kDa intramitochondrial "mature" form of StAR had a half-life of several hours. It was fi rst suggested that importation of StAR into mitochondria formed "contact points" between the OMM and IMM, permitting cholesterol to fl ow down a chemical concentration gradient ( 136,199 ), and others concluded that the 30 kDa "mature" StAR is the biologically active moiety, functioning as a cholesterol shuttle in the intramembranous space lost activity ( 213 ). Thus, the activity of StAR on the OMM requires an acid-induced disruption of hydrogen bonds and a consequent conformational change in StAR to permit it to bind and release cholesterol.…”

“…StAR has a hydrophobic pocket that binds one molecule of cholesterol ( 207,213 ). The interaction of StAR with the OMM involves conformational changes (213)(214)(215)(216)(217)(218) that are needed for it to accept and discharge cholesterol molecules.…”

“…The interaction of StAR with the OMM involves conformational changes (213)(214)(215)(216)(217)(218) that are needed for it to accept and discharge cholesterol molecules. StAR can transfer cholesterol between membranes in vitro ( 219 ), suggesting that other protein molecules are hydrogen bonds, permitting the C-helix to swing open and closed, governing access of cholesterol to the sterolbinding pocket.…”

Les protéines à domaine START, des trafiquants intracellulaires de lipides

“…L'interaction entre l'hélice α4 du domaine START et les têtes protonées des phospholipides de la membrane externe mitochondriale change la conformation de la protéine et ouvre ainsi le couvercle formé par l'hélice α4, donnant accès à la cavité amphiphile du domaine START. En accord avec un rôle crucial de la dynamique de l'hélice α4, des mutants de STARD1 possédant des ponts di-sulfures bloquant la mobilité de l'hélice α4 n'ont plus la capacité de transporter le cholestérol, alors que la réduction de ces ponts di-sulfures restaure le transport [36]. L'import de STARD1 dans la mitochondrie ne serait donc que secondaire à son action et terminerait probablement la stéroïdogenèse.…”