Conformational Flexibility in Crystal Structures of Human 11β-Hydroxysteroid Dehydrogenase Type I Provide Insights into Glucocorticoid Interconversion and Enzyme Regulation

Abstract

Human 11␤-hydroxysteroid dehydrogenase type I (11␤-HSD1) is an ER-localized membrane protein that catalyzes the interconversion of cortisone and cortisol. In adipose tissue, excessive cortisol production through 11␤-HSD1 activity has been implicated in the pathogenesis of type II diabetes and obesity. We report here biophysical, kinetic, mutagenesis, and structural data on two ternary complexes of 11␤-HSD1. The combined results reveal flexible active site interactions relevant to glucocorticoid recognition and demonstrate how four 11␤-HSD1 C termini converge to form an as yet uncharacterized tetramerization motif. A C-terminal Pro-Cys motif is localized at the center of the tetramer and forms reversible enzyme disulfides that alter enzyme activity. Conformational flexibility at the tetramerization interface is coupled to structural changes at the enzyme active site suggesting how the central Pro-Cys motif may regulate enzyme activity. Together, the crystallographic and biophysical data provide a structural framework for understanding 11␤-HSD1 activities and will ultimately facilitate the development of specific inhibitors.

Steroid substrate-induced epimerase mechanism in the active site of the human 11&946;-hydroxysteroid dehydrogenase type 1

“…As expected, no 18 O enrichment was detected in 7α-hydroxy-DHEA or 7β-hydroxy-DHEA after incubation of 7-oxo-DHEA in the presence of NADPH. Both 7α-hydroxy-DHEA and 7β-hydroxy-DHEA were oxidized into 7-oxo-DHEA in NADP + -fortified incubations without 18 O incorporation.…”

“…Tyr 183 is located proximal to the bound cofactor within the human 11β-HSD1 active site and is responsible for the reducing activity 18,19 . This is shown in Fig.…”

“…Both 7α-hydroxy-DHEA and 7β-hydroxy-DHEA were oxidized into 7-oxo-DHEA in NADP + -fortified incubations without 18 O incorporation. As expected from the mechanism proposed above, the reduction of 7-oxoEpiA by NADPH produced both 7α-hydroxy-EpiA and 7β-hydroxy-EpiA containing no 18 O.…”

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

“…The methods for cloning, expression and initial purification for crystallization trials of 11 -HSD1 followed the method described by Hosfield et al (2005) with some adaptation. These are briefly described as follows.…”

“…Diffraction data were indexed, integrated and scaled using HKL-2000 (Otwinowski & Minor, 1997) and the structures were solved by molecular replacement with MOLREP (Vagin & Teplyakov, 2010) in the CCP4 program suite using PDB entry 1xu9 (Hosfield et al, 2005) as a model. The models were refined iteratively using the graphics programs QUANTA (Accelrys, San Diego, California, USA) and REFMAC5 (Murshudov et al, 2011).…”