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

  1. de las Rivas, M. 7
  2. Coelho, H. 148
  3. Diniz, A. 8
  4. Lira-Navarrete, E. 2
  5. Compañón, I. 3
  6. Jiménez-Barbero, J. 146
  7. Schjoldager, K.T. 2
  8. Bennett, E.P. 2
  9. Vakhrushev, S.Y. 2
  10. Clausen, H. 2
  11. Corzana, F. 3
  12. Marcelo, F. 8
  13. Hurtado-Guerrero, R. 57
  1. 1 CIC bioGUNE, Bizkaia Technology Park, Building 801A, Derio, Spain
  2. 2 University of Copenhagen
    info

    University of Copenhagen

    Copenhague, Dinamarca

    ROR https://ror.org/035b05819

  3. 3 Universidad de La Rioja
    info

    Universidad de La Rioja

    Logroño, España

    ROR https://ror.org/0553yr311

  4. 4 Universidad del País Vasco/Euskal Herriko Unibertsitatea
    info

    Universidad del País Vasco/Euskal Herriko Unibertsitatea

    Lejona, España

    ROR https://ror.org/000xsnr85

  5. 5 Fundación ARAID, Zaragoza, Spain
  6. 6 Ikerbasque, Basque Foundation for Science, Maria Diaz de Haro 13, Bilbao, Spain
  7. 7 Universidad de Zaragoza
    info

    Universidad de Zaragoza

    Zaragoza, España

    ROR https://ror.org/012a91z28

  8. 8 Universidade Nova de Lisboa
    info

    Universidade Nova de Lisboa

    Lisboa, Portugal

    ROR https://ror.org/02xankh89

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Revista:
Chemistry - A European Journal

ISSN: 0947-6539

Año de publicación: 2018

Volumen: 24

Número: 33

Páginas: 8382-8392

Tipo: Artículo

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DOI: 10.1002/CHEM.201800701 SCOPUS: 2-s2.0-85048624453 GOOGLE SCHOLAR

Otras publicaciones en: Chemistry - A European Journal

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Resumen

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. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim