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Title: Structure and dynamics of an α-fucosidase reveal a mechanism for highly efficient IgG transfucosylation

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. In this work, 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.

Authors:
 [1]; ORCiD logo [2];  [2];  [1]; ORCiD logo [2]; ORCiD logo [1];  [2];  [2];  [2]; ORCiD logo [1]
  1. Univ. of Maryland, Baltimore, MD (United States)
  2. Univ. of Maryland, College Park, MD (United States)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES)
OSTI Identifier:
1736274
Grant/Contract Number:  
AC02-06CH11357; AC02-76SF00515; P41GM103393; T32 AI095190; R01 GM080374; R01 GM127578; R21AI154232
Resource Type:
Accepted Manuscript
Journal Name:
Nature Communications
Additional Journal Information:
Journal Volume: 11; Journal Issue: 1; Journal ID: ISSN 2041-1723
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; enzyme mechanisms; gylcosides; hydrolases; x-ray crystallography

Citation Formats

Klontz, Erik H., Li, Chao, Kihn, Kyle, Fields, James K., Beckett, Dorothy, Snyder, Greg A., Wintrode, Patrick L., Deredge, Daniel, Wang, Lai-Xi, and Sundberg, Eric J.. Structure and dynamics of an α-fucosidase reveal a mechanism for highly efficient IgG transfucosylation. United States: N. p., 2020. Web. https://doi.org/10.1038/s41467-020-20044-z.
Klontz, Erik H., Li, Chao, Kihn, Kyle, Fields, James K., Beckett, Dorothy, Snyder, Greg A., Wintrode, Patrick L., Deredge, Daniel, Wang, Lai-Xi, & Sundberg, Eric J.. Structure and dynamics of an α-fucosidase reveal a mechanism for highly efficient IgG transfucosylation. United States. https://doi.org/10.1038/s41467-020-20044-z
Klontz, Erik H., Li, Chao, Kihn, Kyle, Fields, James K., Beckett, Dorothy, Snyder, Greg A., Wintrode, Patrick L., Deredge, Daniel, Wang, Lai-Xi, and Sundberg, Eric J.. Fri . "Structure and dynamics of an α-fucosidase reveal a mechanism for highly efficient IgG transfucosylation". United States. https://doi.org/10.1038/s41467-020-20044-z. https://www.osti.gov/servlets/purl/1736274.
@article{osti_1736274,
title = {Structure and dynamics of an α-fucosidase reveal a mechanism for highly efficient IgG transfucosylation},
author = {Klontz, Erik H. and Li, Chao and Kihn, Kyle and Fields, James K. and Beckett, Dorothy and Snyder, Greg A. and Wintrode, Patrick L. and Deredge, Daniel and Wang, Lai-Xi and Sundberg, Eric J.},
abstractNote = {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. In this work, 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.},
doi = {10.1038/s41467-020-20044-z},
journal = {Nature Communications},
number = 1,
volume = 11,
place = {United States},
year = {2020},
month = {12}
}

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Works referenced in this record:

Designer α1,6-Fucosidase Mutants Enable Direct Core Fucosylation of Intact N-Glycopeptides and N-Glycoproteins
journal, October 2017

  • Li, Chao; Zhu, Shilei; Ma, Christopher
  • Journal of the American Chemical Society, Vol. 139, Issue 42
  • DOI: 10.1021/jacs.7b07906

Structural Basis of the Catalytic Reaction Mechanism of Novel 1,2-α-L-Fucosidase from Bifidobacterium bifidum
journal, April 2007

  • Nagae, Masamichi; Tsuchiya, Atsuko; Katayama, Takane
  • Journal of Biological Chemistry, Vol. 282, Issue 25
  • DOI: 10.1074/jbc.M702246200

Lack of Fucose on Human IgG1 N -Linked Oligosaccharide Improves Binding to Human FcγRIII and Antibody-dependent Cellular Toxicity
journal, May 2002

  • Shields, Robert L.; Lai, Jadine; Keck, Rodney
  • Journal of Biological Chemistry, Vol. 277, Issue 30
  • DOI: 10.1074/jbc.M202069200

Analysis of data from the analytical ultracentrifuge by nonlinear least-squares techniques
journal, December 1981


Fcγ receptors as regulators of immune responses
journal, January 2008

  • Nimmerjahn, Falk; Ravetch, Jeffrey V.
  • Nature Reviews Immunology, Vol. 8, Issue 1
  • DOI: 10.1038/nri2206

Probing the Catalytically Essential Residues of the α- l -Fucosidase from the Hyperthermophilic Archaeon Sulfolobus solfataricus
journal, April 2005

  • Cobucci-Ponzano, Beatrice; Mazzone, Marialuisa; Rossi, Mosè
  • Biochemistry, Vol. 44, Issue 16
  • DOI: 10.1021/bi047495f

An Effective Bacterial Fucosidase for Glycoprotein Remodeling
journal, November 2016


Refinement of Macromolecular Structures by the Maximum-Likelihood Method
journal, May 1997

  • Murshudov, G. N.; Vagin, A. A.; Dodson, E. J.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 53, Issue 3
  • DOI: 10.1107/S0907444996012255

CHARMM-GUI: A web-based graphical user interface for CHARMM
journal, March 2008

  • Jo, Sunhwan; Kim, Taehoon; Iyer, Vidyashankara G.
  • Journal of Computational Chemistry, Vol. 29, Issue 11
  • DOI: 10.1002/jcc.20945

Loop engineering of an α-1,3/4-l-fucosidase for improved synthesis of human milk oligosaccharides
journal, August 2018


Fucosylation in prokaryotes and eukaryotes
journal, September 2006

  • Ma, Bing; Simala-Grant, Joanne L.; Taylor, Diane E.
  • Glycobiology, Vol. 16, Issue 12
  • DOI: 10.1093/glycob/cwl040

Glycosynthesis in a waterworld: new insight into the molecular basis of transglycosylation in retaining glycoside hydrolases
journal, March 2015

  • Bissaro, Bastien; Monsan, Pierre; Fauré, Régis
  • Biochemical Journal, Vol. 467, Issue 1
  • DOI: 10.1042/BJ20141412

1,2-α- l -Fucosynthase: A glycosynthase derived from an inverting α-glycosidase with an unusual reaction mechanism
journal, October 2008


Dali server update
journal, April 2016

  • Holm, Liisa; Laakso, Laura M.
  • Nucleic Acids Research, Vol. 44, Issue W1
  • DOI: 10.1093/nar/gkw357

Identification and characterization of a core fucosidase from the bacterium Elizabethkingia meningoseptica
journal, December 2017

  • Li, Tiansheng; Li, Mengjie; Hou, Linlin
  • Journal of Biological Chemistry, Vol. 293, Issue 4
  • DOI: 10.1074/jbc.M117.804252

Modeling of loops in protein structures
journal, January 2000

  • Fiser, András; Do, Richard Kinh Gian; Šali, Andrej
  • Protein Science, Vol. 9, Issue 9
  • DOI: 10.1110/ps.9.9.1753

Directed Evolution of the α- l -Fucosidase from Thermotoga maritima into an α- l -Transfucosidase
journal, January 2007

  • Osanjo, George; Dion, Michel; Drone, Jullien
  • Biochemistry, Vol. 46, Issue 4
  • DOI: 10.1021/bi061444w

A critical review of the role of Fc gamma receptor polymorphisms in the response to monoclonal antibodies in cancer
journal, January 2013

  • Mellor, James D.; Brown, Michael P.; Irving, Helen R.
  • Journal of Hematology & Oncology, Vol. 6, Issue 1
  • DOI: 10.1186/1756-8722-6-1

Features and development of Coot
journal, March 2010

  • Emsley, P.; Lohkamp, B.; Scott, W. G.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4
  • DOI: 10.1107/S0907444910007493

CHARMM-GUI Input Generator for NAMD, GROMACS, AMBER, OpenMM, and CHARMM/OpenMM Simulations Using the CHARMM36 Additive Force Field
journal, December 2015

  • Lee, Jumin; Cheng, Xi; Swails, Jason M.
  • Journal of Chemical Theory and Computation, Vol. 12, Issue 1
  • DOI: 10.1021/acs.jctc.5b00935

1,3-1,4-α-l-Fucosynthase That Specifically Introduces Lewis a/x Antigens into Type-1/2 Chains
journal, March 2012

  • Sakurama, Haruko; Fushinobu, Shinya; Hidaka, Masafumi
  • Journal of Biological Chemistry, Vol. 287, Issue 20
  • DOI: 10.1074/jbc.M111.333781

Crystal Structure of Thermotoga maritima α-l-Fucosidase : INSIGHTS INTO THE CATALYTIC MECHANISM AND THE MOLECULAR BASIS FOR FUCOSIDOSIS
journal, January 2004

  • Sulzenbacher, Gerlind; Bignon, Christophe; Nishimura, Takeshi
  • Journal of Biological Chemistry, Vol. 279, Issue 13
  • DOI: 10.1074/jbc.M313783200

Molecular Cloning and Characterization of Bifidobacterium bifidum 1,2-α-l-Fucosidase (AfcA), a Novel Inverting Glycosidase (Glycoside Hydrolase Family 95)
journal, August 2004


PHENIX: a comprehensive Python-based system for macromolecular structure solution
journal, January 2010

  • Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 213-221
  • DOI: 10.1107/S0907444909052925

Comparison of simple potential functions for simulating liquid water
journal, July 1983

  • Jorgensen, William L.; Chandrasekhar, Jayaraman; Madura, Jeffry D.
  • The Journal of Chemical Physics, Vol. 79, Issue 2
  • DOI: 10.1063/1.445869

β-Glycosyl Azides as Substrates for α-Glycosynthases: Preparation of Efficient α-L-Fucosynthases
journal, October 2009


Unmasking Fucosylation: from Cell Adhesion to Immune System Regulation and Diseases
journal, May 2018


Identification of Essential Residues of Human α- l -Fucosidase and Tests of Its Mechanism
journal, January 2009

  • Liu, Sheng-Wen; Chen, Chao-Sheng; Chang, Shih-Shen
  • Biochemistry, Vol. 48, Issue 1
  • DOI: 10.1021/bi801529t

Structure and Substrate Specificity of a Eukaryotic Fucosidase from Fusarium graminearum
journal, August 2014

  • Cao, Hongnan; Walton, Jonathan D.; Brumm, Phil
  • Journal of Biological Chemistry, Vol. 289, Issue 37
  • DOI: 10.1074/jbc.M114.583286

Utilization of Natural Fucosylated Oligosaccharides by Three Novel α-l-Fucosidases from a Probiotic Lactobacillus casei Strain
journal, November 2010

  • Rodríguez-Díaz, Jesús; Monedero, Vicente; Yebra, María J.
  • Applied and Environmental Microbiology, Vol. 77, Issue 2
  • DOI: 10.1128/AEM.01906-10

Mutagenesis of Glycosidases
journal, June 1999


Molecular replacement with MOLREP
journal, December 2009

  • Vagin, Alexei; Teplyakov, Alexei
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 1
  • DOI: 10.1107/S0907444909042589

Analysis of the Reaction Coordinate of α- l -Fucosidases: A Combined Structural and Quantum Mechanical Approach
journal, February 2010

  • Lammerts van Bueren, Alicia; Fayers-Kerr, Jennifer; Luo, Bo
  • Journal of the American Chemical Society, Vol. 132, Issue 6
  • DOI: 10.1021/ja908908q

Site-specific immobilization of endoglycosidases for streamlined chemoenzymatic glycan remodeling of antibodies
journal, March 2018


Differences in the Substrate Specificities and Active-Site Structures of Two α- L -Fucosidases (Glycoside Hydrolase Family 29) from Bacteroides thetaiotaomicron
journal, May 2012

  • Sakurama, Haruko; Tsutsumi, Erika; Ashida, Hisashi
  • Bioscience, Biotechnology, and Biochemistry, Vol. 76, Issue 5
  • DOI: 10.1271/bbb.111004

Identification of the Catalytic Nucleophile of the Family 29 α-L-Fucosidase from Thermotoga maritima through Trapping of a Covalent Glycosyl-Enzyme Intermediate and Mutagenesis
journal, September 2003

  • Tarling, Chris A.; He, Shouming; Sulzenbacher, Gerlind
  • Journal of Biological Chemistry, Vol. 278, Issue 48
  • DOI: 10.1074/jbc.M306610200

Identifying the Catalytic Acid/Base in GH29 α- l -Fucosidase Subfamilies
journal, August 2013

  • Shaikh, F. Aidha; Lammerts van Bueren, Alicia; Davies, Gideon J.
  • Biochemistry, Vol. 52, Issue 34
  • DOI: 10.1021/bi400183q

Structural basis for the recognition of complex-type N-glycans by Endoglycosidase S
journal, May 2018


MDTraj: A Modern Open Library for the Analysis of Molecular Dynamics Trajectories
journal, October 2015

  • McGibbon, Robert T.; Beauchamp, Kyle A.; Harrigan, Matthew P.
  • Biophysical Journal, Vol. 109, Issue 8
  • DOI: 10.1016/j.bpj.2015.08.015

EndoS2 is a unique and conserved enzyme of serotype M49 group A Streptococcus that hydrolyses N-linked glycans on IgG and α1-acid glycoprotein
journal, September 2013

  • Sjögren, Jonathan; Struwe, Weston B.; Cosgrave, Eoin F. J.
  • Biochemical Journal, Vol. 455, Issue 1
  • DOI: 10.1042/BJ20130126

OpenMM 7: Rapid development of high performance algorithms for molecular dynamics
journal, July 2017


VMD: Visual molecular dynamics
journal, February 1996


The structure of a glycoside hydrolase 29 family member from a rumen bacterium reveals unique, dual carbohydrate-binding domains
journal, September 2016

  • Summers, Emma L.; Moon, Christina D.; Atua, Renee
  • Acta Crystallographica Section F Structural Biology Communications, Vol. 72, Issue 10
  • DOI: 10.1107/S2053230X16014072