3GXM

Crystal structure of acid-beta-glucosidase at pH 4.5, phosphate crystallization condition

3GXM の概要

• エントリーDOI: 10.2210/pdb3gxm/pdb
• 関連するPDBエントリー: 3GXF 3GXI
• 分子名称: Glucosylceramidase, 2-acetamido-2-deoxy-beta-D-glucopyranose, SULFATE ION, ... (4 entities in total)
• 機能のキーワード: hydrolase, alternative initiation, disease mutation, disulfide bond, gaucher disease, glycoprotein, glycosidase, ichthyosis, lipid metabolism, lysosome, membrane, sphingolipid metabolism
• 由来する生物種: Homo sapiens (human)
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 226135.27

構造登録者

Lieberman, R.L. (登録日: 2009-04-02, 公開日: 2009-05-05, 最終更新日: 2020-07-29)

主引用文献

Lieberman, R.L.,D'aquino, J.A.,Ringe, D.,Petsko, G.A.
Effects of pH and iminosugar pharmacological chaperones on lysosomal glycosidase structure and stability.
Biochemistry, 48:4816-4827, 2009

PubMed Abstract: Human lysosomal enzymes acid-beta-glucosidase (GCase) and acid-alpha-galactosidase (alpha-Gal A) hydrolyze the sphingolipids glucosyl- and globotriaosylceramide, respectively, and mutations in these enzymes lead to the lipid metabolism disorders Gaucher and Fabry disease, respectively. We have investigated the structure and stability of GCase and alpha-Gal A in a neutral-pH environment reflective of the endoplasmic reticulum and an acidic-pH environment reflective of the lysosome. These details are important for the development of pharmacological chaperone therapy for Gaucher and Fabry disease, in which small molecules bind mutant enzymes in the ER to enable the mutant enzyme to meet quality control requirements for lysosomal trafficking. We report crystal structures of apo GCase at pH 4.5, at pH 5.5, and in complex with the pharmacological chaperone isofagomine (IFG) at pH 7.5. We also present thermostability analysis of GCase at pH 7.4 and 5.2 using differential scanning calorimetry. We compare our results with analogous experiments using alpha-Gal A and the chaperone 1-deoxygalactonijirimycin (DGJ), including the first structure of alpha-Gal A with DGJ. Both GCase and alpha-Gal A are more stable at lysosomal pH with and without their respective iminosugars bound, and notably, the stability of the GCase-IFG complex is pH sensitive. We show that the conformations of the active site loops in GCase are sensitive to ligand binding but not pH, whereas analogous galactose- or DGJ-dependent conformational changes in alpha-Gal A are not seen. Thermodynamic parameters obtained from alpha-Gal A unfolding indicate two-state, van't Hoff unfolding in the absence of the iminosugar at neutral and lysosomal pH, and non-two-state unfolding in the presence of DGJ. Taken together, these results provide insight into how GCase and alpha-Gal A are thermodynamically stabilized by iminosugars and suggest strategies for the development of new pharmacological chaperones for lysosomal storage disorders.

PubMed: 19374450
DOI: 10.1021/bi9002265

実験手法

X-RAY DIFFRACTION (2.2 Å)