Incorrect Folding of Steroidogenic Acute Regulatory Protein (StAR) in Congenital Lipoid Adrenal Hyperplasia

• Published in: Biochemistry (Easton) Vol. 37; no. 27; pp. 9768 - 9775
• Main Authors: Bose, Himangshu S, Baldwin, Michael A, Miller, Walter L
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 07.07.1998
• Subjects: Adrenal Hyperplasia, Congenital - genetics; Adrenal Hyperplasia, Congenital - metabolism; Circular Dichroism; Escherichia coli - genetics; Guanidine - pharmacology; Hot Temperature; Humans; Mutagenesis, Site-Directed; Phosphoproteins - chemistry; Phosphoproteins - genetics; Phosphoproteins - isolation & purification; Protein Denaturation - drug effects; Protein Folding; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Spectroscopy, Fourier Transform Infrared; Urea - pharmacology
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi980588a

Steroidogenic acute regulatory protein (StAR) rapidly stimulates the movement of cholesterol into adrenal and gonadal mitochondria to mediate the acute steroidogenic response; StAR mutations cause potentially lethal congenital lipoid adrenal hyperplasia (lipoid CAH). Bacterially expressed wild-type StAR and four amino acid replacement/deletion mutants that cause lipoid CAH were purified to apparent homogeneity. Sedimentation equilibrium ultracentrifugation showed that all five proteins were monomeric and fit a globular protein model of the correct molecular mass. Circular dichroism (CD) spectra of both the wild-type and mutants showed minima near 208 and 222 nm, confirming the presence of substantial α-helical structure. However, subtle differences in the CD signals of the wild-type and mutants in the far-UV and stronger differences in near-UV indicated differences in protein folding. The amide I and II bands in the 1400−1700 cm-1 region of Fourier transform infrared spectra showed that the proteins fell into two groups. The wild-type and a partially active conservative mutant were predominantly α-helical with some intramolecular β-sheet. By contrast, three mutants that lost charged residues retained much of their α-helical structure, but also tended to form intermolecular β-sheets. Urea at 2.0 or 4.0 M had less effect on the CD spectrum of the wild-type than of the mutants, particularly those having lost a charged residue; 50 mM guanidinium hydrochloride did not alter the CD spectrum of the wild-type, but elicited dramatic changes to the secondary structure in all four mutants. Despite this, thermal melting curves of the mutant proteins in 50 mM guanidinium hydrochloride showed surprising stability, even exceeding that of the wild-type protein. These data suggest that the StAR amino acid replacement mutants that cause lipoid CAH are inactive because of fairly gross errors in protein folding, probably due to the loss of salt bridges that stabilize the tertiary structure.