Structural basis for the synthesis of the core 1 structure by C1GalT1

The glycosyltransferase C1GalT1 directs a key step in protein O-glycosylation important for the expression of the cancer-associated Tn and T antigens. Here, the authors provide molecular insights into the function of C1GalT1 by solving the crystal structure of the Drosophila enzyme-substrate complex. C1GalT1 is an essential inverting glycosyltransferase responsible for synthesizing the core 1 structure, a common precursor for mucin-type O-glycans found in many glycoproteins. To date, the structure of C1GalT1 and the details of substrate recognition and catalysis remain unknown. Through biophysical and cellular studies, including X-ray crystallography of C1GalT1 complexed to a glycopeptide, we report that C1GalT1 is an obligate GT-A fold dimer that follows a S(N)2 mechanism. The binding of the glycopeptides to the enzyme is mainly driven by the GalNAc moiety while the peptide sequence provides optimal kinetic and binding parameters. Interestingly, to achieve glycosylation, C1GalT1 recognizes a high-energy conformation of the alpha-GalNAc-Thr linkage, negligibly populated in solution. By imposing this 3D-arrangement on that fragment, characteristic of alpha-GalNAc-Ser peptides, C1GalT1 ensures broad glycosylation of both acceptor substrates. These findings illustrate a structural and mechanistic blueprint to explain glycosylation of multiple acceptor substrates, extending the repertoire of mechanisms adopted by glycosyltransferases.

Automated summary

This study provides molecular insights into the function of C1GalT1, a glycosyltransferase that plays a key role in protein O-glycosylation. The crystal structure of the Drosophila enzyme-substrate complex reveals that C1GalT1 is an obligate GT-A fold dimer that follows a S(N)2 mechanism. The binding of glycopeptides to C1GalT1 is mainly driven by the GalNAc moiety, and the peptide sequence provides optimal kinetic and binding parameters. C1GalT1 recognizes a high-energy conformation of the alpha-GalNAc-Thr linkage to achieve glycosylation. These findings contribute to our understanding of glycosylation mechanisms adopted by glycosyltransferases.