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

7 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

Keywords

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

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    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