The crystal structure of murine 11β-hydroxysteroid dehydrogenase 1: an important therapeutic target for diabetes

Zhang, J.; Osslund, Timothy D.; Plant, Matthew; Clogston, Christi L.; Nybo, Rebecca; Xiong, Fei; Delaney, John; Jordan, Steven R.

doi:10.1107/s0108767305091956

Cited by 13 publications

(22 citation statements)

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“…Attempts to express the active-site mutant K187N in the human F278E background also resulted in a protein that would not express in a soluble form in the recombinant E. coli system. Because the K187 residue is known to be involved in cofactor binding, 14,20,24,28 these results again suggest that the interaction with NADP(H) is required for correct folding and stability, at least in E. coli. This is supported by the observation that high-level expression of human 11b-HSD1 in a soluble form in bacteria requires addition of an inhibitor compound, 36 for example, the steroid analogue carbenoxolone used in these studies, which also presumably requires prior cofactor binding.…”

Section: Discussionmentioning

confidence: 59%

“…Crystals were grown as per Materials and Methods with the data collection and refinements statistics given in Table III. Each monomer in the guinea pig F278E structure had the same overall Rossman fold as the other published 11b-HSD1 structures 14,24,28 and had a quaternary assembly of a tetramer made up of a dimer-of-dimers [ Fig. 6(A)].…”

Section: Crystal Structure Of Guinea Pig F278ementioning

confidence: 94%

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Mutations of key hydrophobic surface residues of 11β‐hydroxysteroid dehydrogenase type 1 increase solubility and monodispersity in a bacterial expression system

et al. 2009

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11b-Hydroxysteroid dehydrogenase type 1 (11b-HSD1) is a key enzyme in the conversion of cortisone to the functional glucocorticoid hormone cortisol. This activation has been implicated in several human disorders, notably the metabolic syndrome where 11b-HSD1 has been identified as a novel target for potential therapeutic drugs. Recent crystal structures have revealed the presence of a pronounced hydrophobic surface patch lying on two helices at the C-terminus. The physiological significance of this region has been attributed to facilitating substrate access by allowing interactions with the endoplasmic reticulum membrane. Here, we report that single mutations that alter the hydrophobicity of this patch (I275E, L266E, F278E, and L279E in the human enzyme and I275E, Y266E, F278E, and L279E in the guinea pig enzyme) result in greatly increased yields of soluble protein on expression in E. coli. Kinetic analyses of both reductase and dehydrogenase reactions indicate that the F278E mutant has unaltered K m values for steroids and an unaltered or increased k cat . Analytical ultracentrifugation shows that this mutation also decreases aggregation of both the human and guinea pig enzymes, resulting in greater monodispersity. One of the mutants (guinea pig F278E) has proven easy to crystallize and has been shown to have a virtually identical structure to that previously reported for the wild-type enzyme. The human F278E enzyme is shown to be a suitable background for analyzing the effects of naturally occurring mutations (R137C, K187N) on enzyme activity and stability. Hence, the F278E mutants should be useful for many future biochemical and biophysical studies of the enzyme.

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Section: Discussionmentioning

confidence: 59%

Section: Crystal Structure Of Guinea Pig F278ementioning

confidence: 94%

Mutations of key hydrophobic surface residues of 11β‐hydroxysteroid dehydrogenase type 1 increase solubility and monodispersity in a bacterial expression system

et al. 2009

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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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The near‐symmetry of proteins

Bonjack-Shterengartz

Avnir

2015

Proteins

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The majority of protein oligomers form clusters which are nearly symmetric. Understanding of that imperfection, its origins, and perhaps also its advantages requires the conversion of the currently used vague qualitative descriptive language of the near-symmetry into an accurate quantitative measure that will allow to answer questions such as: "What is the degree of symmetry deviation of the protein?," "how do these deviations compare within a family of proteins?," and so on. We developed quantitative methods to answer this type of questions, which are capable of analyzing the whole protein, its backbone or selected portions of it, down to comparison of symmetry-related specific amino-acids, and which are capable of visualizing the various levels of symmetry deviations in the form of symmetry maps. We have applied these methods on an extensive list of homomers and heteromers and found that apparently all proteins never reach perfect symmetry. Strikingly, even homomeric protein clusters are never ideally symmetric. We also found that the main burden of symmetry distortion is on the amino-acids near the symmetry axis; that it is mainly the more hydrophilic amino-acids that take place in symmetry-distortive interactions; and more. The remarkable ability of heteromers to preserve near-symmetry, despite the different sequences, was also shown and analyzed. The comprehensive literature on the suggested advantages symmetric oligomerizations raises a yet-unsolved key question: If symmetry is so advantageous, why do proteins stop shy of perfect symmetry? Some tentative answers to be tested in further studies are suggested in a concluding outlook.

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The crystal structure of murine 11β-hydroxysteroid dehydrogenase 1: an important therapeutic target for diabetes

Cited by 13 publications

References 0 publications

Mutations of key hydrophobic surface residues of 11β‐hydroxysteroid dehydrogenase type 1 increase solubility and monodispersity in a bacterial expression system

Mutations of key hydrophobic surface residues of 11β‐hydroxysteroid dehydrogenase type 1 increase solubility and monodispersity in a bacterial expression system

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

The near‐symmetry of proteins

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