Steroidogenesis depends on the delivery of free cholesterol to the inner mitochondrial membrane by StAR (steroidogenic acute regulatory protein). Mutations in the StAR gene leads to proteins with limited cholesterol-binding capacity. This gives rise to the accumulation of cytoplasmic cholesterol, a deficit in steroid hormone production and to the medical condition of lipoid congenital adrenal hyperplasia. A detailed understanding of the mechanism of the specific binding of free cholesterol by StAR would be a critical asset in understanding the molecular origin of this disease. Previous studies have led to the proposal that the C-terminal α-helix 4 of StAR was undergoing a folding/unfolding transition. This transition is thought to gate the cholesterol-binding site. Moreover, a conserved salt bridge (Glu169–Arg188) in the cholesterol-binding site is also proposed to be critical to the binding process. Interestingly, some of the documented clinical mutations occur at this salt bridge (E169G, E169K and R188C) and in the C-terminal α-helix 4 (L275P). In the present study, using rationalized mutagenesis, activity assays, CD, thermodynamic studies and molecular modelling, we characterized the α-helix 4 mutations L271N and L275P, as well as the salt bridge double mutant E169M/R188M. The results provide experimental validation for the gating mechanism of the cholesterol-binding site by the C-terminal α-helix and the importance of the salt bridge in the binding mechanism. Altogether, our results offer a molecular framework for understanding the impact of clinical mutations on the reduction of the binding affinity of StAR for free cholesterol.
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April 2009
Research Article|
January 21 2009
The mechanism of specific binding of free cholesterol by the steroidogenic acute regulatory protein: evidence for a role of the C-terminal α-helix in the gating of the binding site
Alireza Roostaee;
Alireza Roostaee
1
*Département de Biochimie, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, Québec, Canada, J1H 5N4
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Élie Barbar;
Élie Barbar
1
*Département de Biochimie, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, Québec, Canada, J1H 5N4
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Pierre Lavigne;
Pierre Lavigne
§Département de Pharmacologie, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, Québec, Canada, J1H 5N4
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Jean-Guy Lehoux
Jean-Guy Lehoux
2
*Département de Biochimie, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, Québec, Canada, J1H 5N4
2To whom correspondence should be addressed (email Jean-Guy.LeHoux@USherbrooke.ca).
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Biosci Rep (2009) 29 (2): 89–101.
Article history
Received:
August 15 2008
Accepted:
August 26 2008
Accepted Manuscript online:
August 26 2008
Citation
Alireza Roostaee, Élie Barbar, Pierre Lavigne, Jean-Guy Lehoux; The mechanism of specific binding of free cholesterol by the steroidogenic acute regulatory protein: evidence for a role of the C-terminal α-helix in the gating of the binding site. Biosci Rep 1 April 2009; 29 (2): 89–101. doi: https://doi.org/10.1042/BSR20080111
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