Structural Basis for Peptide Substrate Specificities of Glycosyltransferase GalNAc-T2

Abstract

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.

Structure-based prediction of HDAC6 substrates validated by enzymatic assay reveals determinants of promiscuity and detects new potential substrates

“…Structure-based methods can complement sequence-based methods, particularly in cases of non-canonical motifs 22 , 24 , as we have previously shown for PTM enzymes using the Rosetta FlexPepBind protocol 12 , 25 , 26 . This approach assumes that the ability of the substrate local peptide sequence to bind in a catalysis-competent conformation is a main determinant of enzyme selectivity, and thus the binding energy of such enzyme–substrate complex structures can be taken as a proxy for substrate activity.…”

“…Hidden Markov Models 17 and naive Bayes 18 ), but such approaches depend on considerable amounts of data 19 – 21 . Moreover, enzyme substrate patterns may not always adequately be depicted by a sequence-based description, like in the case of O-glycosylation 22 or HIV-1 protease substrates 23 .…”

Quick and Accurate Estimates of Mutation Effects on Transition-State Stabilization of Enzymes from Molecular Simulations with Restrained Transition States

“…For more details regarding the method used to obtain this transition-state model, see our previous work or consult the documentation for ATESA at atesa.readthedocs.io. For our model of the GalNAc-T2, we followed the methodology laid out in ref based on PDB IDs: 2FFU and 4D0Z . We excluded the noncatalytic lectin domain, which is not believed to interact directly with the substrates during the reaction .…”

Research overview on the genetic mechanism underlying the biosynthesis of polysaccharide in tuber plants

“…This mechanism suggests that the nucleophilic attack and departure of the leaving group take place on the same side of the sugar group, resulting in the formation of a brief oxocarbenium-like transition state. Subsequently, the acceptor C-O glycosidic link is formed and the C-O bond between the sugar group and the phosphate group is cleaved, so concluding the catalytic process of configuration retention ( Mahajan et al, 2021 ). Ultimately, additional experimental evidence is required to determine whether the configuration-preserving GTs may be executed using various catalytic methods.…”