TY - JOUR
T1 - Structural Basis for Peptide Substrate Specificities of Glycosyltransferase GalNAc-T2
AU - Mahajan, Sai Pooja
AU - Srinivasan, Yashes
AU - Labonte, Jason W.
AU - Delisa, Matthew P.
AU - Gray, Jeffrey J.
N1 - Publisher Copyright:
© 2021 American Chemical Society.
PY - 2021/3/5
Y1 - 2021/3/5
N2 - The polypeptide N-acetylgalactosaminyl transferase (GalNAc-T) enzyme family initiates O-linked mucin-type glycosylation. The family constitutes 20 isoenzymes in humans. 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 nonglycosylatable peptides with a probability of 96.5% (ROC-AUC score), a balanced accuracy of 85.5%, and a false positive rate of 7.3%. 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 suboptimal 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.
AB - The polypeptide N-acetylgalactosaminyl transferase (GalNAc-T) enzyme family initiates O-linked mucin-type glycosylation. The family constitutes 20 isoenzymes in humans. 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 nonglycosylatable peptides with a probability of 96.5% (ROC-AUC score), a balanced accuracy of 85.5%, and a false positive rate of 7.3%. 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 suboptimal 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.
KW - GalNAcT
KW - O-linked glycosylation
KW - computational specificity prediction
KW - enzyme specificity
KW - glycosyltransferases
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U2 - 10.1021/acscatal.0c04609
DO - 10.1021/acscatal.0c04609
M3 - Article
C2 - 34322281
AN - SCOPUS:85102116618
SN - 2155-5435
VL - 11
SP - 2977
EP - 2991
JO - ACS Catalysis
JF - ACS Catalysis
IS - 5
ER -