title: Crystal Structure and Functional Analyses of the Lectin Domain of Glucosidase II: Insights into Oligomannose Recognition.
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.
authors.affiliation: †Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, United States.
authors.forename: Linda J
authors.initials: LJ
authors.lastname: Olson
authors.affiliation: ‡Laboratory of Glycobiology, Fundación Instituto Leloir and Instituto de Investigaciones Bioquímicas de Buenos Aires-CONICET, Buenos Aires, Argentina.
authors.forename: Ramiro
authors.initials: R
authors.lastname: Orsi
authors.affiliation: †Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, United States.
authors.forename: Francis C
authors.initials: FC
authors.lastname: Peterson
authors.affiliation: ‡Laboratory of Glycobiology, Fundación Instituto Leloir and Instituto de Investigaciones Bioquímicas de Buenos Aires-CONICET, Buenos Aires, Argentina.
authors.forename: Armando J
authors.initials: AJ
authors.lastname: Parodi
authors.affiliation: †Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, United States.
authors.forename: Jung-Ja P
authors.initials: JJ
authors.lastname: Kim
authors.affiliation: ‡Laboratory of Glycobiology, Fundación Instituto Leloir and Instituto de Investigaciones Bioquímicas de Buenos Aires-CONICET, Buenos Aires, Argentina.|§School of Sciences, University of Buenos Aires, Buenos Aires, Argentina.
authors.forename: Cecilia
authors.initials: C
authors.lastname: D'Alessio
authors.affiliation: †Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, United States.
authors.forename: Nancy M
authors.initials: NM
authors.lastname: Dahms
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018360:Crystallography, X-Ray; D006801:Humans; D037102:Lectins / Q000378:metabolism*; D008358:Mannose / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017527:Receptor, IGF Type 2 / Q000378:metabolism; D012568:Schizosaccharomyces / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D000520:alpha-Glucosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D037102:Lectins; D021122:Protein Subunits; D017527:Receptor, IGF Type 2; C034251:4-nitrophenyl-alpha-glucosidase; D000520:alpha-Glucosidases; D008358:Mannose