Faculty Collaboration Database

Crystal Structure and Functional Analyses of the Lectin Domain of Glucosidase II: Insights into Oligomannose Recognition. Biochemistry 2015 Jul 07;54(26):4097-111

Date

06/11/2015

Pubmed ID

26062005

Pubmed Central ID

PMC6202027

DOI

10.1021/acs.biochem.5b00256

Scopus ID

2-s2.0-84936769044 (requires institutional sign-in at Scopus site)   14 Citations

Abstract

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 GIIα is enhanced by the mannose 6-phosphate receptor (MPR) homology domain (MRH domain) of its β subunit, we now report a 1.6 Å resolution crystal structure of the MRH domain of GIIβ 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 GIIβ, 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 GIIβ 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 GIIβ or GIIα subunit to modulate its effect on GII activity.

Author List

Olson LJ, Orsi R, Peterson FC, Parodi AJ, Kim JJ, D'Alessio C, Dahms NM

Authors

Nancy M. Dahms PhD Professor in the Biochemistry department at Medical College of Wisconsin
Linda J. Olson PhD Assistant Professor in the Pharmacology and Toxicology department at Medical College of Wisconsin
Francis C. Peterson PhD Professor in the Biochemistry department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Amino Acid Sequence
Animals
Crystallography, X-Ray
Humans
Lectins
Mannose
Models, Molecular
Molecular Sequence Data
Point Mutation
Protein Structure, Tertiary
Protein Subunits
Receptor, IGF Type 2
Schizosaccharomyces
Sequence Alignment
alpha-Glucosidases