Toward the NMR structure of StAR

doi:10.1016/j.mce.2008.12.007

Molecular and Cellular Endocrinology

Volume 300, Issues 1–2, 5 March 2009, Pages 89-93

https://doi.org/10.1016/j.mce.2008.12.007 Get rights and content

Abstract

The steroidogenic acute regulatory (StAR) protein plays a crucial role in steroidogenesis, as it accelerates the transport of cholesterol to the inner mitochondrial membrane where the cytochrome P450scc enzyme is located. Mutations in the StAR gene can lead to lipoid congenital adrenal hyperplasia (LCAH), a disease that is fatal if not treated with hormone replacement therapy. Solving the structure of StAR is an important aspect of understanding LCAH. Point mutations or truncations in the StAR gene produce a partial to non-functional protein that hinders the StAR-induced delivery of cholesterol to the mitochondria during an acute hormonal stimulation of steroidogenic cells. So far, homology modeling, structure-based thermodynamics and biophysical studies have allowed us to propose the existence of an open state of StAR where the C-terminal α-helix 4 undergoes partial unfolding. This may act as a gating mechanism to the cholesterol binding site. Once bound, cholesterol leads to the stabilization and the refolding of α-helix 4, and eventually to the interaction with an import complex at the surface of the mitochondria. Though the current homology models have proven useful in understanding StAR function, only the full determination of the 3D structure of the apo- and holo-states will further validate this two-state model. In this context, we have used solution-state nuclear magnetic resonance (NMR) and obtained high-resolution ¹H–¹⁵N-HSQC spectra of StAR in its apo- and holo-states at physiological pH. Both spectra displayed well-dispersed resonances. However, key differences are observed on the spectra which indicate that both states have stable but slightly different tertiary structures. In conjunction with the binding/activity assays and biophysical methods, this original NMR data constitutes another structural step into the validation of the two-state model and the three-dimensional structure of StAR.

Section snippets

Steroidogenesis

The first step in steroidogenesis is the conversion of precursor cholesterol to pregnenolone by the action of the cytochrome P450 side chain cleavage enzyme (P450scc, product of the CYP11A1 gene) present at the inner mitochonrial membrane (IMM) (Brownie et al., 1972, Privalle et al., 1983). For a long time, the activity of P450scc was thought to be the rate-limiting step in steroidogenesis. However, it was found that cycloheximide blocked the translocation of cholesterol from the outer

The START domain

StAR has an N-terminal mitochondrial import sequence (Arakane et al., 1998, Wang et al., 1998), and a steroidogenic acute regulatory protein-related lipid transfer (START) domain that plays a role in the binding and transfer of cholesterol (Tsujishita and Hurley, 2000, Petrescu et al., 2001). The START domain is part of a superfamily of proteins found in prokaryotes, animals and plants (Iyer et al., 2001, Schrick et al., 2004, Alpy and Tomasetto, 2005). Proteins with a START domain possess a

StAR mutations in LCAH

More than 30 mutations in the StAR gene lead to non-functional proteins that cause lipoid congenital adrenal hyperplasia (LCAH) (Bose et al., 1996, Bose et al., 2000). Mutated StAR lacks the ability to bind free cholesterol and to transfer it to the OMM. During an acute hormonal stimulation, this causes an accumulation of cholesterol in the cytoplasm and an engorgement of the cell with lipid droplets, thereby impairing steroid synthesis in LCAH patients. According to the molecular models of

Role of α-helix 4

To understand the precise mechanism of StAR and its role in steroidogenesis, it is necessary to elucidate its structure as well as its interaction with cholesterol. In a previous study, we characterized the thermodynamics of N-62 StAR (StAR without its mitochondrial import sequence) and determined that cholesterol bound the protein with a 1:1 ratio with an apparent K_D of 3 × 10⁻⁸ (Roostaee et al., 2008). These results are in agreement with the current structural model illustrating the cholesterol

The clinical mutation L275P and the role of the salt bridge

In another series of experiments (Roostaee et al., in press), the role of StAR's salt bridge and α-helix 4 in the binding mechanism of free cholesterol was investigated with more detail using structure-based mutagenesis. The hydrophobic cluster formed by α-helix 4 in relation to the bound cholesterol (Fig. 2) was studied. For instance, the clinical mutation L275P had a lesser α-helicity, it retained only a fraction of the cholesterol binding and had less steroidogenic activity in comparison to

NMR spectroscopy of StAR

Thus far, the structural validation of the two-state model for StAR action relied on biochemical and thermodynamic analyses. However, this methodology is limited to macroscopic parameters, and a structural approach at the atomic level is required to confirm the homology models of the free and bound states. Crystallization of StAR was previously attempted (Tsujishita and Hurley, 2000), but due to aggregation problems the task did not succeed. And so, we focused our efforts to study StAR by

Cholesterol release/delivery mechanism

StAR acts at the OMM (Bose et al., 2002) and has an increased capacity to bind synthetic membranes at very low (artificial) pH (Christensen et al., 2001). It was hypothesized that the acidic polar head groups of membrane phospholipids would cause StAR to become a molten globule which in turn becomes the active form of the protein (Bose et al., 1999). Acidic pH is a particularly effective method for artificially generating molten globule states for structural studies (Kumar et al., 1995,

Conclusion

In conclusion, the homology modeling, structure-based mutagenesis and biophysical studies have provided us with a theoretical framework for understanding the mechanism of StAR action in steroidogenesis and to rationalize the impact of mutations regarding the accumulation of cholesterol in the clinical cases of LCAH. Thus far biochemical, cellular, structural and thermodynamic studies have brought a substantial experimental validation of our two-state model. However, to gain deeper insights into

Acknowledgment

This work was supported by a grant to Jean-Guy LeHoux from the Canadian Institute of Health Research, MT-10983.

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ReviewToward the NMR structure of StAR

Abstract

Section snippets

Steroidogenesis

The START domain

StAR mutations in LCAH

Role of α-helix 4

The clinical mutation L275P and the role of the salt bridge

NMR spectroscopy of StAR

Cholesterol release/delivery mechanism

Conclusion

Acknowledgment

J. Biol. Chem.

Adv. Protein Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

J. Lipid Res.

Mol. Cell. Endocrinol.

J. Biol. Chem.

J. Biol. Chem.

Adv. Protein Chem.

J. Biol. Chem.

Mol. Cell. Endocrinol.

J. Biol. Chem.

Energized, polarized, and actively respiring mitochondria are required for acute Leydig cell steroidogenesis

Endocrinology

Review
Toward the NMR structure of StAR