The Crystal Structure of Guinea Pig 11β-Hydroxysteroid Dehydrogenase Type 1 Provides a Model for Enzyme-Lipid Bilayer Interactions

Ogg, D.; Elleby, Björn; Norström, Carina; Stefansson, Karin; Abrahmsén, Lars; Oppermann, U.; Svensson, Stefan

doi:10.1074/jbc.m412463200

Cited by 49 publications

(61 citation statements)

References 34 publications

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“…In this model Cys-175 is found at the N terminus of helix F oriented perpendicular to and in close vicinity to the lipid membrane surface. This region was shown to form a short-chain dehydrogenase dimerization interface (64,67). Because of this structural feature, efficient cross-linking between RPE65 and RDH5 may occur only with the monomeric form of retinol dehydrogenase.…”

Section: Discussionmentioning

confidence: 99%

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: Discussionmentioning

confidence: 99%

Importance of Membrane Structural Integrity for RPE65 Retinoid Isomerization Activity

Golczak

Kiser

Lodowski

et al. 2010

Journal of Biological Chemistry

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“…11β-HSD1 is a dimeric enzyme, and although a mutant K187N homodimer would be expected to be inactive, the effect of heterodimer formation between WT and mutant subunits (as could occur in heterozygous humans, such as case B) was uncertain. The two subunits of 11β-HSD1 are closely intertwined, facilitating a close structural interplay between the monomers (4,5). Thus, the K187N mutation in one subunit possibly could negatively affect the activity of an adjacent WT subunit.…”

Section: Discussionmentioning

confidence: 99%

“…11β-HSD1 is a member of the short-chain dehydrogenase reductase (SDR) superfamily of enzymes and naturally exists as a homodimer (2)(3)(4)(5), although some studies have suggested that it also may function as a homotetramer (6). Unusually for an SDR enzyme, 11β-HSD1 has an N-terminal membrane anchor (7,8), with a small portion of the N-terminal segment of the enzyme present in the cytosol.…”

mentioning

confidence: 99%

“…Unusually for an SDR enzyme, 11β-HSD1 has an N-terminal membrane anchor (7,8), with a small portion of the N-terminal segment of the enzyme present in the cytosol. The glycosylated catalytic C-terminal domain exists in the lumen of the endoplasmic reticulum (8) and putatively dips into the membrane lipid bilayer to facilitate access to steroid substrates (4,5). The reaction direction that 11β-HSD1 catalyzes is determined by the relative abundance of NADP + and NADPH (9,10).…”

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confidence: 99%

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Cortisone-reductase deficiency associated with heterozygous mutations in 11β-hydroxysteroid dehydrogenase type 1

Lawson¹,

Walker

Lavery

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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In peripheral target tissues, levels of active glucocorticoid hormones are controlled by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), a dimeric enzyme that catalyzes the reduction of cortisone to cortisol within the endoplasmic reticulum. Loss of this activity results in a disorder termed cortisone reductase deficiency (CRD), typified by increased cortisol clearance and androgen excess. To date, only mutations in H6PD, which encodes an enzyme supplying cofactor for the reaction, have been identified as the cause of disease. Here we examined the HSD11B1 gene in two cases presenting with biochemical features indicative of a milder form of CRD in whom the H6PD gene was normal. Novel heterozygous mutations (R137C or K187N) were found in the coding sequence of HSD11B1. The R137C mutation disrupts salt bridges at the subunit interface of the 11β-HSD1 dimer, whereas K187N affects a key active site residue. On expression of the mutants in bacterial and mammalian cells, activity was either abolished (K187N) or greatly reduced (R137C). Expression of either mutant in a bacterial system greatly reduced the yield of soluble protein, suggesting that both mutations interfere with subunit folding or dimer assembly. Simultaneous expression of mutant and WT 11β-HSD1 in bacterial or mammalian cells, to simulate the heterozygous condition, indicated a marked suppressive effect of the mutants on both the yield and activity of 11β-HSD1 dimers. Thus, these heterozygous mutations in the HSD11B1 gene have a dominant negative effect on the formation of functional dimers and explain the genetic cause of CRD in these patients.

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“…Although we used a similar cloning and purification procedure, we were unable to displace CHAPS with our adamantane compounds by soaking, cocrystallization or other commonly used complexation procedures. While this is puzzling, another researcher working with guinea pig 11 -HSD1 also reported problems in ligand exchange (Ogg et al, 2005). In this case, the protein was incubated during purification with NADP + and ligand (BVT.4584).…”

Section: Discussionmentioning

confidence: 99%

On-column ligand exchange for structure-based drug design: a case study with human 11β-hydroxysteroid dehydrogenase type 1

Qin¹,

Judge

Longenecker

et al. 2012

Acta Cryst Sect F

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Successfully forming ligand-protein complexes with specific compounds can be a significant challenge in supporting structure-based drug design for a given protein target. In this respect, an on-column ligand-and detergent-exchange method was developed to obtain ligand-protein complexes of an adamantane series of compounds with 11 -hydroxysteroid dehydrogenase type 1 (11 -HSD1) after a variety of other complexation methods had failed. This report describes the on-column exchange method and an unexpected byproduct of the method in which artificial trimers were observed in the structures.

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The Crystal Structure of Guinea Pig 11β-Hydroxysteroid Dehydrogenase Type 1 Provides a Model for Enzyme-Lipid Bilayer Interactions

Cited by 49 publications

References 34 publications

Importance of Membrane Structural Integrity for RPE65 Retinoid Isomerization Activity

Importance of Membrane Structural Integrity for RPE65 Retinoid Isomerization Activity

Cortisone-reductase deficiency associated with heterozygous mutations in 11β-hydroxysteroid dehydrogenase type 1

On-column ligand exchange for structure-based drug design: a case study with human 11β-hydroxysteroid dehydrogenase type 1

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