Structural Basis for Catalysis and Inhibition ofN-Glycan Processing Class I α1,2-Mannosidases

François Vallée,Khanita Karaveg,Annette Herscovics,Kelley W Moremen,P Lynne Howell

doi:10.1074/jbc.m006927200

Abstract

Endoplasmic reticulum (ER) class I alpha1,2-mannosidase (also known as ER alpha-mannosidase I) is a critical enzyme in the maturation of N-linked oligosaccharides and ER-associated degradation. Trimming of a single mannose residue acts as a signal to target misfolded glycoproteins for degradation by the proteasome. Crystal structures of the catalytic domain of human ER class I alpha1,2-mannosidase have been determined both in the presence and absence of the potent inhibitors kifunensine and 1-deoxymannojirimycin. Both inhibitors bind to the protein at the bottom of the active-site cavity, with the essential calcium ion coordinating the O-2' and O-3' hydroxyls and stabilizing the six-membered rings of both inhibitors in a (1)C(4) conformation. This is the first direct evidence of the role of the calcium ion. The lack of major conformational changes upon inhibitor binding and structural comparisons with the yeast alpha1, 2-mannosidase enzyme-product complex suggest that this class of inverting enzymes has a novel catalytic mechanism. The structures also provide insight into the specificity of this class of enzymes and provide a blueprint for the future design of novel inhibitors that prevent degradation of misfolded proteins in genetic diseases.

Highlights

The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
Crystal structures of the catalytic domain of human Endoplasmic reticulum (ER) class I ␣1,2mannosidase have been determined both in the presence and absence of the potent inhibitors kifunensine and 1-deoxymannojirimycin
N-Glycan formation begins with the transfer of a preformed oligosaccharide precursor, Glc3Man9GlcNAc2, to the nascent polypeptide chain. ␣-Glucosidases and ␣-mannosidases in the ER trim this oligosaccharide precursor to primarily Man8GlcNAc2, while subsequent transport of the glycoprotein into the Golgi apparatus allows further trimming of the remaining ␣1,2-linked mannose residues

Summary

Introduction

The atomic coordinates and structure factors (code 1FM1, 1FO2, and 1FO3) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). Both inhibitors bind to the protein at the bottom of the active-site cavity, with the essential calcium ion coordinating the O-2؅ and O-3؅ hydroxyls and stabilizing the six-membered rings of both inhibitors in a 1C4 conformation.

Results

Conclusion