Structure and dynamics of an alpha-fucosidase reveal a mechanism for highly efficient IgG transfucosylation

• Type of Material: Article
• Authors: Erik H. Klontz, University of Maryland School of Medicine; Chao Li, University of Maryland School of Medicine; Kyle Kihn, University of Maryland School of Medicine; James K. Fields, University of Maryland School of Medicine; Dorothy Beckett, University of Maryland School of Medicine; Greg A. Snyder, University of Maryland School of Medicine; Patrick L. Wintrode, University of Maryland School of Medicine; Daniel Deredge, University of Maryland School of Medicine; Lai-Xi Wang, University of Maryland School of Medicine; Eric Sundberg, Emory University
• Language: English
• Date: 2020-12-04
• Publisher: NATURE RESEARCH
• Publication Version: Final Published Version
• Copyright Statement: © The Author(s) 2020.
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1038/s41467-020-20044-z
• Title of Journal or Parent Work: NATURE COMMUNICATIONS
• Volume: 11
• Issue: 1
• Start Page: 6204
• End Page: 6204
• Grant/Funding Information: This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. Additionally, Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (including P41GM103393). This work is also supported in part by the University of Maryland Baltimore, School of Pharmacy Mass Spectrometry Center (SOP1841-IQB2014), as well as NIH grants T32 AI095190 (E.K.), R01 GM080374 (L.-X.W.), R01 GM127578, and R21AI154232 (E.S.). The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
• Keywords: Multidisciplinary Sciences; MOLECULAR-BASIS; SOFTWARE NEWS; ESSENTIAL RESIDUES; PROTEIN; IDENTIFICATION; GLYCOSIDASE; 1,2-ALPHA-L-FUCOSIDASE; THERMOTOGA-MARITIMA; Science & Technology - Other Topics; BINDING; Science & Technology; STREPTOCOCCUS
• Research Categories: Health Sciences, Immunology; Biology, Virology; Biology, Microbiology

Abstract

Fucosylation is important for the function of many proteins with biotechnical and medical applications. Alpha-fucosidases comprise a large enzyme family that recognizes fucosylated substrates with diverse α-linkages on these proteins. Lactobacillus casei produces an α-fucosidase, called AlfC, with specificity towards α(1,6)-fucose, the only linkage found in human N-glycan core fucosylation. AlfC and certain point mutants thereof have been used to add and remove fucose from monoclonal antibody N-glycans, with significant impacts on their effector functions. Despite the potential uses for AlfC, little is known about its mechanism. Here, we present crystal structures of AlfC, combined with mutational and kinetic analyses, hydrogen–deuterium exchange mass spectrometry, molecular dynamic simulations, and transfucosylation experiments to define the molecular mechanisms of the activities of AlfC and its transfucosidase mutants. Our results indicate that AlfC creates an aromatic subsite adjacent to the active site that specifically accommodates GlcNAc in α(1,6)-linkages, suggest that enzymatic activity is controlled by distinct open and closed conformations of an active-site loop, with certain mutations shifting the equilibrium towards open conformations to promote transfucosylation over hydrolysis, and provide a potentially generalizable framework for the rational creation of AlfC transfucosidase mutants.