The structure of the membrane protein squalene-hopene cyclase at 2.0 å resolution

Wendt, K. Ulrich; Lenhart, Alexander; Schulz, Georg E.

doi:10.1006/jmbi.1998.2470

Cited by 205 publications

(264 citation statements)

References 46 publications

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“…Thus, we speculate that formation of the large solvent channel observed in the crystal was necessary to accommodate the associated detergent molecules. Indeed, similar packing arrangements have been observed in crystals of monotopic membrane proteins, such as prostaglandin H 2 synthases and squalene-hopene cyclase, that were crystallized in the presence of detergent (55).…”

Section: Discussionsupporting

confidence: 61%

Importance of Membrane Structural Integrity for RPE65 Retinoid Isomerization Activity

Golczak

Kiser

Lodowski

et al. 2010

Journal of Biological Chemistry

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Regeneration of visual chromophore in the vertebrate visual cycle involves the retinal pigment epithelium-specific protein RPE65, the key enzyme catalyzing the cleavage and isomerization of all-trans-retinyl fatty acid esters to 11-cis-retinol. Although RPE65 has no predicted membrane spanning domains, this protein predominantly associates with microsomal fractions isolated from bovine retinal pigment epithelium (RPE). We have re-examined the nature of RPE65 interactions with native microsomal membranes by using extraction and phase separation experiments. We observe that hydrophobic interactions are the dominant forces that promote RPE65 association with these membranes. These results are consistent with the crystallographic model of RPE65, which features a large lipophilic surface that surrounds the entrance to the catalytic site of this enzyme and likely interacts with the hydrophobic core of the endoplasmic reticulum membrane. Moreover, we report a critical role for phospholipid membranes in preserving the retinoid isomerization activity and physical properties of RPE65. Isomerase activity measured in bovine RPE was highly sensitive to phospholipase A 2 treatment, but the observed decline in 11-cis-retinol production did not directly reflect inhibition by products of lipid hydrolysis. Instead, a direct correlation between the kinetics of phospholipid hydrolysis and retinoid isomerization suggests that the lipid membrane structure is critical for RPE65 enzymatic activity. We also provide evidence that RPE65 operates in a multiprotein complex with retinol dehydrogenase 5 and retinal G protein-coupled receptor in RPE microsomes. Modifications in the phospholipid environment affecting interactions with these protein components may be responsible for the alterations in retinoid metabolism observed in phospholipid-depleted RPE microsomes. Thus, our results indicate that the enzymatic activity of native RPE65 strongly depends on its membrane binding and phospholipid environment.In vertebrates, the perception of light is preceded by a quantum event in which a photon of light hits the 11-cis-retinylidene chromophore of rhodopsin in the retina. Photoisomerization of this chromophore to its all-trans isomer results in activation of rhodopsin triggering a cascade of signal transduction events (summarized in Ref. 1). This process simultaneously leads to loss of photoreceptor light sensitivity making continuous regeneration of 11-cis-retinal a requirement to sustain vision. The visual (retinoid) cycle is the fundamental series of reactions responsible for regeneration of the visual chromophore that occurs in photoreceptors and the retinal pigment epithelium (RPE) 4 (summarized in Ref.2). The key component of this enzymatic cycle is the RPE-specific microsomal membrane protein known as RPE65 (3-5). The importance of RPE65 in the retinoid cycle is underscored by the metabolic dysfunction found in Rpe65 Ϫ/Ϫ mice, which is characterized by complete lack of 11-cis-retinoid production accompanied by accumulation of the all-tr...

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Section: Discussionsupporting

confidence: 61%

Importance of Membrane Structural Integrity for RPE65 Retinoid Isomerization Activity

Golczak

Kiser

Lodowski

et al. 2010

Journal of Biological Chemistry

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“…However, our structural model precludes this possibility, because two helices of the four-helix bundle (and their associated iron ligands) would fall on the opposite side of the membrane. Instead, we propose that Coq7 associates with the membrane as an interfacial integral membrane protein, similar to prostaglandin synthase (29) and squalene cyclase (30). In these proteins a "plateau" of hydrophobic residues interacts with one leaflet of the membrane bilayer.…”

Section: Coq7 Is a Di-iron Carboxylate Protein 33298mentioning

confidence: 93%

A New Member of the Family of Di-iron Carboxylate Proteins

Stenmark

Grünler

Mattsson

et al. 2001

Journal of Biological Chemistry

124

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Ubiquinone (UQ) is an essential cofactor for respiratory metabolism. In yeast, mutation of the COQ7 gene results in the absence of UQ biosynthesis and demonstrates a role for this gene in the step leading to the hydroxylation of 5-demethoxyubiquinone. Intriguingly, the disruption of the corresponding gene in Caenorhabditis elegans, clk-1, results in a prolonged life span and a slowing of development. Because of the pleiotropic effect of this disruption, the small size of the protein, and the lack of obvious homology to other known hydroxylases, it has been suggested that Coq7 may be a regulatory or structural component in UQ biosynthesis, rather than acting as the hydroxylase per se. Here we identify Coq7 as belonging to a family of a di-iron containing oxidases/hydroxylases based on a conserved sequence motif for the iron ligands, supporting a direct function of Coq7 as a hydroxylase. We have cloned COQ7 from Pseudomonas aeruginosa and Thiobacillus ferrooxidans and show that indeed this gene complements an Escherichia coli mutant that lacks an unrelated 5-demethoxyubiquinone hydroxylase. Based on the similarities to other well studied di-iron carboxylate proteins, we propose a structural model for Coq7 as an interfacial integral membrane protein.

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“…However, modeling studies have predicted Coq7 to be an interfacial inner mitochondrial membrane protein [61,62]. Interfacial membrane proteins, such as prostaglandin synthase [63] and squalene cyclase [64], are embedded in the membrane via interaction with only one leaflet of the bilayer. To distinguish between a peripheral and an interfacial membrane association for Coq7, salt extraction analyses (with 0.5 M KCl, 1.0 M KCl, or 0.5 M NaCl) were performed on sonicated mitoplasts prepared as described above.…”

Section: Coq2 Is Resistant To Salt Extraction While Other Coq Polypepmentioning

confidence: 99%

Coenzyme Q supplementation or Over-Expression of the Yeast Coq8 Putative Kinase Stabilizes Multi-Subunit Coq Polypeptide Complexes in Yeast Coq Null Mutants

Xie

Allan

et al. 2013

Free Radical Biology and Medicine

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The structure of the membrane protein squalene-hopene cyclase at 2.0 å resolution

Cited by 205 publications

References 46 publications

Importance of Membrane Structural Integrity for RPE65 Retinoid Isomerization Activity

Importance of Membrane Structural Integrity for RPE65 Retinoid Isomerization Activity

A New Member of the Family of Di-iron Carboxylate Proteins

Coenzyme Q supplementation or Over-Expression of the Yeast Coq8 Putative Kinase Stabilizes Multi-Subunit Coq Polypeptide Complexes in Yeast Coq Null Mutants

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