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The structure of the colorectal cancer-associated enzyme GalNAc-T12 reveals how nonconserved residues dictate its function.

ABSTRACT: Polypeptide N-acetylgalactosaminyl transferases (GalNAc-Ts) initiate mucin type O-glycosylation by catalyzing the transfer of N-acetylgalactosamine (GalNAc) to Ser or Thr on a protein substrate. Inactive and partially active variants of the isoenzyme GalNAc-T12 are present in subsets of patients with colorectal cancer, and several of these variants alter nonconserved residues with unknown functions. While previous biochemical studies have demonstrated that GalNAc-T12 selects for peptide and glycopeptide substrates through unique interactions with its catalytic and lectin domains, the molecular basis for this distinct substrate selectivity remains elusive. Here we examine the molecular basis of the activity and substrate selectivity of GalNAc-T12. The X-ray crystal structure of GalNAc-T12 in complex with a di-glycosylated peptide substrate reveals how a nonconserved GalNAc binding pocket in the GalNAc-T12 catalytic domain dictates its unique substrate selectivity. In addition, the structure provides insight into how colorectal cancer mutations disrupt the activity of GalNAc-T12 and illustrates how the rules dictating GalNAc-T12 function are distinct from those for other GalNAc-Ts.

SUBMITTER: Fernandez AJ

PROVIDER: S-EPMC6789641 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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The structure of the colorectal cancer-associated enzyme GalNAc-T12 reveals how nonconserved residues dictate its function.

Fernandez Amy J AJ Daniel Earnest James Paul EJP Mahajan Sai Pooja SP Gray Jeffrey J JJ Gerken Thomas A TA Tabak Lawrence A LA Samara Nadine L NL

Proceedings of the National Academy of Sciences of the United States of America 20190923 41

Polypeptide N-acetylgalactosaminyl transferases (GalNAc-Ts) initiate mucin type O-glycosylation by catalyzing the transfer of N-acetylgalactosamine (GalNAc) to Ser or Thr on a protein substrate. Inactive and partially active variants of the isoenzyme GalNAc-T12 are present in subsets of patients with colorectal cancer, and several of these variants alter nonconserved residues with unknown functions. While previous biochemical studies have demonstrated that GalNAc-T12 selects ...[more]

PMID: 31548401

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Project description:In two-component signal transduction, response regulator proteins contain the catalytic machinery for their own covalent phosphorylation and can catalyze phosphotransfer from a partner sensor kinase or autophosphorylate using various small molecule phosphodonors. Although response regulator autophosphorylation is physiologically relevant and a powerful experimental tool, the kinetic determinants of the autophosphorylation reaction and how those determinants might vary for different response regulators and phosphodonors are largely unknown. We characterized the autophosphorylation kinetics of 21 variants of the model response regulator Escherichia coli CheY that contained substitutions primarily at nonconserved active site positions D + 2 (CheY residue 59) and T + 2 (CheY residue 89), two residues C-terminal to conserved D57 and T87, respectively. Overall, the CheY variants exhibited a >10(5)-fold range of rate constants (kphos/KS) for reaction with phosphoramidate, acetyl phosphate, or monophosphoimidazole, with the great majority of rates enhanced versus that of wild-type CheY. Although phosphodonor preference varied substantially, nearly all the CheY variants reacted faster with phosphoramidate than acetyl phosphate. Correlation between the increased positive charge of the D + 2 and T + 2 side chains and faster rates indicated electrostatic interactions are a kinetic determinant. Moreover, sensitivities of rate constants to ionic strength indicated that both long-range and localized electrostatic interactions influence autophosphorylation kinetics. The increased nonpolar surface area of the D + 2 and T + 2 side chains also correlated with an enhanced autophosphorylation rate, especially for reaction with phosphoramidate and monophosphoimidazole. Computer docking suggested that highly accelerated monophosphoimidazole autophosphorylation rates for CheY variants with a tyrosine at position T + 2 likely reflect structural mimicry of phosphotransfer from the sensor kinase histidyl phosphate.

Dataset Information

The structure of the colorectal cancer-associated enzyme GalNAc-T12 reveals how nonconserved residues dictate its function.

Publications

The structure of the colorectal cancer-associated enzyme GalNAc-T12 reveals how nonconserved residues dictate its function.

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