Structural Analysis of a GalNAc-T2 Mutant Reveals an Induced-Fit Catalytic Mechanism for GalNAc-Ts

Matilde de las Rivas, Helena Coelho, Ana Diniz, Erandi Lira-Navarrete, Ismael Compañón, Jesús Jiménez-Barbero, Katrine T. Schjoldager, Eric P. Bennett, Sergey Y. Vakhrushev, Henrik Clausen, Francisco Corzana, Filipa Marcelo, Ramon Hurtado-Guerrero

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

The family of polypeptide N-acetylgalactosamine (GalNAc) transferases (GalNAc-Ts) orchestrates the initiating step of mucin-type protein O-glycosylation by transfer of GalNAc moieties to serine and threonine residues in proteins. Deficiencies and dysregulation of GalNAc-T isoenzymes are related to different diseases. Recently, it has been demonstrated that an inactive GalNAc-T2 mutant (F104S), which is not located at the active site, induces low levels of high-density lipoprotein cholesterol (HDL-C) in humans. Herein, the molecular basis for F104S mutant inactivation has been deciphered. Saturation transfer difference NMR spectroscopy experiments demonstrate that the mutation induces loss of binding to peptide substrates. Analysis of the crystal structure of the F104S mutant bound to UDP-GalNAc (UDP=uridine diphosphate), combined with molecular dynamics (MD) simulations, has revealed that the flexible loop is disordered and displays larger conformational changes in the mutant enzyme than that in the wild-type (WT) enzyme. 19F NMR spectroscopy experiments reveal that the WT enzyme only reaches the active state in the presence of UDP-GalNAc, which provides compelling evidence that GalNAc-T2 adopts a UDP-GalNAc-dependent induced-fit mechanism. The F104S mutation precludes the enzyme from achieving the active conformation and concomitantly binding peptide substrates. This study provides new insights into the catalytic mechanism of the large family of GalNAc-Ts and how these enzymes orchestrate protein O-glycosylation.

Original languageEnglish
Pages (from-to)8382-8392
Number of pages11
JournalChemistry - A European Journal
Volume24
Issue number33
DOIs
Publication statusPublished - 12 Jun 2018

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Uridine Diphosphate
Structural analysis
Enzymes
Glycosylation
Proteins
Peptides
Nuclear magnetic resonance spectroscopy
Isoenzymes
Acetylgalactosamine
Lipoproteins
Polypeptides
Cholesterol
Mucins
Substrates
Threonine
Serine
HDL Cholesterol
Conformations
Molecular dynamics
Crystal structure

Keywords

  • enzymes
  • glycosylation
  • mutagenesis
  • oligomers
  • structure–activity relationships

Cite this

de las Rivas, M., Coelho, H., Diniz, A., Lira-Navarrete, E., Compañón, I., Jiménez-Barbero, J., ... Hurtado-Guerrero, R. (2018). Structural Analysis of a GalNAc-T2 Mutant Reveals an Induced-Fit Catalytic Mechanism for GalNAc-Ts. Chemistry - A European Journal, 24(33), 8382-8392. https://doi.org/10.1002/chem.201800701
de las Rivas, Matilde ; Coelho, Helena ; Diniz, Ana ; Lira-Navarrete, Erandi ; Compañón, Ismael ; Jiménez-Barbero, Jesús ; Schjoldager, Katrine T. ; Bennett, Eric P. ; Vakhrushev, Sergey Y. ; Clausen, Henrik ; Corzana, Francisco ; Marcelo, Filipa ; Hurtado-Guerrero, Ramon. / Structural Analysis of a GalNAc-T2 Mutant Reveals an Induced-Fit Catalytic Mechanism for GalNAc-Ts. In: Chemistry - A European Journal. 2018 ; Vol. 24, No. 33. pp. 8382-8392.
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abstract = "The family of polypeptide N-acetylgalactosamine (GalNAc) transferases (GalNAc-Ts) orchestrates the initiating step of mucin-type protein O-glycosylation by transfer of GalNAc moieties to serine and threonine residues in proteins. Deficiencies and dysregulation of GalNAc-T isoenzymes are related to different diseases. Recently, it has been demonstrated that an inactive GalNAc-T2 mutant (F104S), which is not located at the active site, induces low levels of high-density lipoprotein cholesterol (HDL-C) in humans. Herein, the molecular basis for F104S mutant inactivation has been deciphered. Saturation transfer difference NMR spectroscopy experiments demonstrate that the mutation induces loss of binding to peptide substrates. Analysis of the crystal structure of the F104S mutant bound to UDP-GalNAc (UDP=uridine diphosphate), combined with molecular dynamics (MD) simulations, has revealed that the flexible loop is disordered and displays larger conformational changes in the mutant enzyme than that in the wild-type (WT) enzyme. 19F NMR spectroscopy experiments reveal that the WT enzyme only reaches the active state in the presence of UDP-GalNAc, which provides compelling evidence that GalNAc-T2 adopts a UDP-GalNAc-dependent induced-fit mechanism. The F104S mutation precludes the enzyme from achieving the active conformation and concomitantly binding peptide substrates. This study provides new insights into the catalytic mechanism of the large family of GalNAc-Ts and how these enzymes orchestrate protein O-glycosylation.",
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author = "{de las Rivas}, Matilde and Helena Coelho and Ana Diniz and Erandi Lira-Navarrete and Ismael Compa{\~n}{\'o}n and Jes{\'u}s Jim{\'e}nez-Barbero and Schjoldager, {Katrine T.} and Bennett, {Eric P.} and Vakhrushev, {Sergey Y.} and Henrik Clausen and Francisco Corzana and Filipa Marcelo and Ramon Hurtado-Guerrero",
note = "info:eu-repo/grantAgreement/FCT/5876/147258/PT# We thank the Ministerio de Economia y Competitividad (CTQ2013-44367-C2-2-P and BFU2016-75633-P to R.H-G., CTQ2015-67727-R to F.C., CTQ2015-64597-C2-1P to J.J-B). F.M. thanks FCT-Portugal for the IF project (IF/00780/2015) and UCIBIO funding UID/Multi/04378/2013 cofinanced by the FEDER (POCI-01-0145-FEDER-007728). The NMR spectrometers are part of PTNMR supported by project no. 022161 (co-financed by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC). H.C. thanks the Lundbeck Foundation and the Danish National Research Foundation (DNRF107). E.L-N. acknowledges her postdoctoral EMBO fellowship ALTF 1553-2015 cofunded by the European Commission (LTFCO-FUND2013, GA-2013-609409) and Marie Curie Actions. H.C. and J.J-B. thank the EU for the TOLLerant project. R.H-G. thanks the Agencia Aragonesa para la Investigacion y Desarrollo (ARAID) and the Diputacion General de Aragon (DGA, B89) for financial support. Research leading to these results has also received funding from the FP7 (2007-2013) under BioStruct-X (grant agreement no. 283570 and BIOSTRUCTX_5186). We thank synchrotron radiation source DIAMOND (Oxford) and beamline I04 (number of experiment MX10121-19). We would also like to thank to Prof. Tom Gerken for his valuable comments on our manuscript.",
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de las Rivas, M, Coelho, H, Diniz, A, Lira-Navarrete, E, Compañón, I, Jiménez-Barbero, J, Schjoldager, KT, Bennett, EP, Vakhrushev, SY, Clausen, H, Corzana, F, Marcelo, F & Hurtado-Guerrero, R 2018, 'Structural Analysis of a GalNAc-T2 Mutant Reveals an Induced-Fit Catalytic Mechanism for GalNAc-Ts', Chemistry - A European Journal, vol. 24, no. 33, pp. 8382-8392. https://doi.org/10.1002/chem.201800701

Structural Analysis of a GalNAc-T2 Mutant Reveals an Induced-Fit Catalytic Mechanism for GalNAc-Ts. / de las Rivas, Matilde; Coelho, Helena; Diniz, Ana; Lira-Navarrete, Erandi; Compañón, Ismael; Jiménez-Barbero, Jesús; Schjoldager, Katrine T.; Bennett, Eric P.; Vakhrushev, Sergey Y.; Clausen, Henrik; Corzana, Francisco; Marcelo, Filipa; Hurtado-Guerrero, Ramon.

In: Chemistry - A European Journal, Vol. 24, No. 33, 12.06.2018, p. 8382-8392.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Structural Analysis of a GalNAc-T2 Mutant Reveals an Induced-Fit Catalytic Mechanism for GalNAc-Ts

AU - de las Rivas, Matilde

AU - Coelho, Helena

AU - Diniz, Ana

AU - Lira-Navarrete, Erandi

AU - Compañón, Ismael

AU - Jiménez-Barbero, Jesús

AU - Schjoldager, Katrine T.

AU - Bennett, Eric P.

AU - Vakhrushev, Sergey Y.

AU - Clausen, Henrik

AU - Corzana, Francisco

AU - Marcelo, Filipa

AU - Hurtado-Guerrero, Ramon

N1 - info:eu-repo/grantAgreement/FCT/5876/147258/PT# We thank the Ministerio de Economia y Competitividad (CTQ2013-44367-C2-2-P and BFU2016-75633-P to R.H-G., CTQ2015-67727-R to F.C., CTQ2015-64597-C2-1P to J.J-B). F.M. thanks FCT-Portugal for the IF project (IF/00780/2015) and UCIBIO funding UID/Multi/04378/2013 cofinanced by the FEDER (POCI-01-0145-FEDER-007728). The NMR spectrometers are part of PTNMR supported by project no. 022161 (co-financed by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC). H.C. thanks the Lundbeck Foundation and the Danish National Research Foundation (DNRF107). E.L-N. acknowledges her postdoctoral EMBO fellowship ALTF 1553-2015 cofunded by the European Commission (LTFCO-FUND2013, GA-2013-609409) and Marie Curie Actions. H.C. and J.J-B. thank the EU for the TOLLerant project. R.H-G. thanks the Agencia Aragonesa para la Investigacion y Desarrollo (ARAID) and the Diputacion General de Aragon (DGA, B89) for financial support. Research leading to these results has also received funding from the FP7 (2007-2013) under BioStruct-X (grant agreement no. 283570 and BIOSTRUCTX_5186). We thank synchrotron radiation source DIAMOND (Oxford) and beamline I04 (number of experiment MX10121-19). We would also like to thank to Prof. Tom Gerken for his valuable comments on our manuscript.

PY - 2018/6/12

Y1 - 2018/6/12

N2 - The family of polypeptide N-acetylgalactosamine (GalNAc) transferases (GalNAc-Ts) orchestrates the initiating step of mucin-type protein O-glycosylation by transfer of GalNAc moieties to serine and threonine residues in proteins. Deficiencies and dysregulation of GalNAc-T isoenzymes are related to different diseases. Recently, it has been demonstrated that an inactive GalNAc-T2 mutant (F104S), which is not located at the active site, induces low levels of high-density lipoprotein cholesterol (HDL-C) in humans. Herein, the molecular basis for F104S mutant inactivation has been deciphered. Saturation transfer difference NMR spectroscopy experiments demonstrate that the mutation induces loss of binding to peptide substrates. Analysis of the crystal structure of the F104S mutant bound to UDP-GalNAc (UDP=uridine diphosphate), combined with molecular dynamics (MD) simulations, has revealed that the flexible loop is disordered and displays larger conformational changes in the mutant enzyme than that in the wild-type (WT) enzyme. 19F NMR spectroscopy experiments reveal that the WT enzyme only reaches the active state in the presence of UDP-GalNAc, which provides compelling evidence that GalNAc-T2 adopts a UDP-GalNAc-dependent induced-fit mechanism. The F104S mutation precludes the enzyme from achieving the active conformation and concomitantly binding peptide substrates. This study provides new insights into the catalytic mechanism of the large family of GalNAc-Ts and how these enzymes orchestrate protein O-glycosylation.

AB - The family of polypeptide N-acetylgalactosamine (GalNAc) transferases (GalNAc-Ts) orchestrates the initiating step of mucin-type protein O-glycosylation by transfer of GalNAc moieties to serine and threonine residues in proteins. Deficiencies and dysregulation of GalNAc-T isoenzymes are related to different diseases. Recently, it has been demonstrated that an inactive GalNAc-T2 mutant (F104S), which is not located at the active site, induces low levels of high-density lipoprotein cholesterol (HDL-C) in humans. Herein, the molecular basis for F104S mutant inactivation has been deciphered. Saturation transfer difference NMR spectroscopy experiments demonstrate that the mutation induces loss of binding to peptide substrates. Analysis of the crystal structure of the F104S mutant bound to UDP-GalNAc (UDP=uridine diphosphate), combined with molecular dynamics (MD) simulations, has revealed that the flexible loop is disordered and displays larger conformational changes in the mutant enzyme than that in the wild-type (WT) enzyme. 19F NMR spectroscopy experiments reveal that the WT enzyme only reaches the active state in the presence of UDP-GalNAc, which provides compelling evidence that GalNAc-T2 adopts a UDP-GalNAc-dependent induced-fit mechanism. The F104S mutation precludes the enzyme from achieving the active conformation and concomitantly binding peptide substrates. This study provides new insights into the catalytic mechanism of the large family of GalNAc-Ts and how these enzymes orchestrate protein O-glycosylation.

KW - enzymes

KW - glycosylation

KW - mutagenesis

KW - oligomers

KW - structure–activity relationships

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EP - 8392

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SN - 1521-3765

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