Cholesterol binding is a prerequisite for the activity of the steroidogenic acute regulatory protein (StAR)

Abstract

Steroidogenesis depends on the delivery of cholesterol from the outer to the inner mitochondrial membrane by StAR (steroidogenic acute regulatory protein). However, the mechanism by which StAR binds to cholesterol and its importance in cholesterol transport are under debate. According to our proposed molecular model, StAR possesses a hydrophobic cavity, which can accommodate one cholesterol molecule. In the bound form, cholesterol interacts with hydrophobic side-chains located in the C-terminal alpha-helix 4, thereby favouring the folding of this helix. To verify this model experimentally, we have characterized the in vitro activity, overall structure, thermodynamic stability and cholesterol-binding affinity of StAR lacking the N-terminal 62 amino acid residues (termed N-62 StAR). This mature form is biologically active and has a well-defined tertiary structure. Addition of cholesterol to N-62 StAR led to an increase in the alpha-helical content and T degrees (melting temperature), indicating the formation of a stable complex. However, the mutation F267Q, which is located in the C-terminal helix interface lining the cholesterol-binding site, reduced the biological activity of StAR. Furthermore, the cholesterol-induced thermodynamic stability and the binding capacity of StAR were significantly diminished in the F267Q mutant. Titration of StAR with cholesterol yielded a 1:1 complex with an apparent K(D) of 3 x 10(-8). These results support our model and indicate that StAR can readily bind to cholesterol with an apparent affinity that commensurates with monomeric cholesterol solubility in water. The proper function of the C-terminal alpha-helix is essential for the binding process.

Aggregation and Amyloid Fibril Formation Induced by Chemical Dimerization of Recombinant Prion Protein in Physiological-like Conditions

“…AP20187-treated rFv-PrP was directly added to cells at a final concentration of 10 M. Staurosporine was used at a concentration of 100 nM to induce apoptosis in control experiments (36). Control aggregates of steroidogenic acute regulatory protein (StAR) protein (27) were obtained with 2 M urea at pH 5.0 and added to cells in the same concentration as rFv-PrP.…”

“…After a washing step with buffer R devoid of ␤-cyclodextrin (Buffer W), the protein was eluted with the same buffer containing 500 mM imidazole and 100 mM GdnHCl (Buffer E). The eluted fractions were concentrated (27) in buffer W. All buffers contained 30 mM imidazole, unless indicated otherwise.…”

“…The temperature dependence of the mean residue ellipticity () at 222 nm was fitted using an in-house non-linear least-squares fitting program (29). To obtain the thermodynamic parameters (T°, ⌬H u 0 (T°), ⌬S u 0 (T°), and ⌬G u 0 (T°)) describing the stability of rFv-PrP structural species, thermal denaturation curves of rFv-PrP were simulated assuming a two-state unfolding mechanism (27)(28)(29). These experiments were performed in triplicates.…”

StAR-related lipid transfer domain protein 5 binds primary bile acids

“…We previously demonstrated the binding of cholesterol to STARD1 using CD ( 48 ) and NMR ( 58 ); essentially, addition of cholesterol led to the thermodynamic stabilization of the START domain of STARD1 and to drastic changes in the chemical shifts of the 1 H-15 N correlations in its HSQC spectrum. However, under the same experimental conditions, addition of cholesterol to STARD5 did not induce noticeable changes in the far-UV CD spectrum, in the thermodynamic stability (shift in the temperature denaturation curve), or in the 1 H-15 N HSQC (see supplementary data).…”

“…·K Ϫ 1 (a typical value for a protein the size of STARD5) as described in Roostae et al ( 48 ). Measurements with the ligand in appropriate buffer were performed at a protein to ligand molar ratio of 1:100, and spectra were taken at a molar ratio of 1:100.…”