Crystal Structure and Functional Analyses of the Lectin Domain of Glucosidase II: Insights into Oligomannose Recognition

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• 作者：Linda J. Olson ; Ramiro Orsi ; Francis C. Peterson ; Armando J. Parodi ; Jung-Ja P. Kim ; Cecilia D鈥橝lessio ; Nancy M. Dahms
• 刊名：Biochemistry
• 出版年：2015
• 出版时间：July 7, 2015
• 年：2015
• 卷：54
• 期：26
• 页码：4097-4111
• 全文大小：1049K
• ISSN：1520-4995

文摘

N-Glycans are modified as part of a quality control mechanism during glycoprotein folding in the endoplasmic reticulum (ER). Glucosidase II (GII) plays a critical role by generating monoglucosylated glycans that are recognized by lectin chaperones, calnexin and calreticulin. To understand how the hydrolytic activity of GII伪 is enhanced by the mannose 6-phosphate receptor (MPR) homology domain (MRH domain) of its 尾 subunit, we now report a 1.6 脜 resolution crystal structure of the MRH domain of GII尾 bound to mannose. A comparison of ligand-bound and unbound structures reveals no major difference in their overall fold, but rather a repositioning of side chains throughout the binding pocket, including Y372. Mutation of Y372 inhibits GII activity, demonstrating an important role for Y372 in regulating GII activity. Comparison of the MRH domains of GII尾, MPRs, and the ER lectin OS-9 identified conserved residues that are critical for the structural integrity and architecture of the carbohydrate binding pocket. As shown by nuclear magnetic resonance spectroscopy, mutations of the primary binding pocket residues and adjacent W409, all of which inhibit the activity of GII both in vitro and in vivo, do not cause a significant change in the overall fold of the GII尾 MRH domain but impact locally the stability of the binding pocket. W409 does not directly contact mannose; rather, its indole ring is stabilized by binding into a hydrophobic pocket of an adjacent crystallographic neighbor. This suggests that W409 interacts with a hydrophobic region of the GII尾 or GII伪 subunit to modulate its effect on GII activity.