PT  - JOURNAL ARTICLE
AU  - Sai Pooja Mahajan
AU  - Yashes Srinivasan
AU  - Jason W. Labonte
AU  - Matthew P. DeLisa
AU  - Jeffrey J. Gray
TI  - Structural basis for peptide substrate specificities of glycosyltransferase GalNAc-T2
AID  - 10.1101/2020.06.25.171371
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.06.25.171371
4099  - http://biorxiv.org/content/early/2020/06/27/2020.06.25.171371.short
4100  - http://biorxiv.org/content/early/2020/06/27/2020.06.25.171371.full
AB  - The polypeptide N-acetylgalactosaminyl transferase (GalNAc-T) enzyme family initiates O-linked mucin-type glycosylation. The family constitutes 20 isozymes in humans—an unusually large number—unique to O-glycosylation. GalNAc-Ts exhibit both redundancy and finely tuned specificity for a wide range of peptide substrates. In this work, we deciphered the sequence and structural motifs that determine the peptide substrate preferences for the GalNAc-T2 isoform. Our approach involved sampling and characterization of peptide–enzyme conformations obtained from Rosetta Monte Carlo-minimization–based flexible docking. We computationally scanned 19 amino acid residues at positions –1 and +1 of an eight-residue peptide substrate, which comprised a dataset of 361 (19×19) peptides with previously characterized experimental GalNAc-T2 glycosylation efficiencies. The calculations recapitulated experimental specificity data, successfully discriminating between glycosylatable and non-glycosylatable peptides with an accuracy of 89% and a ROC-AUC score of 0.965. The glycosylatable peptide substrates viz. peptides with proline, serine, threonine, and alanine at the –1 position of the peptide preferentially exhibited cognate sequon-like conformations. The preference for specific residues at the −1 position of the peptide was regulated by enzyme residues R362, K363, Q364, H365 and W331, which modulate the pocket size and specific enzyme-peptide interactions. For the +1 position of the peptide, enzyme residues K281 and K363 formed gating interactions with aromatics and glutamines at the +1 position of the peptide, leading to modes of peptide-binding sub-optimal for catalysis. Overall, our work revealed enzyme features that lead to the finely tuned specificity observed for a broad range of peptide substrates for the GalNAc-T2 enzyme. We anticipate that the key sequence and structural motifs can be extended to analyze specificities of other isoforms of the GalNAc-T family and can be used to guide design of variants with tailored specificity.Competing Interest StatementThe authors have declared no competing interest.