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Bump-and-hole engineering of human polypeptide N-acetylgalactosamine transferases to dissect their protein substrates and glycosylation sites in cells.

ABSTRACT: Despite the known disease relevance of glycans, the biological function and substrate specificities of individual glycosyltransferases are often ill-defined. Here, we describe a protocol to develop chemical, bioorthogonal reporters for the activity of the GalNAc-T family of glycosyltransferases using a tactic termed bump-and-hole engineering. This allows identification of the protein substrates and glycosylation sites of single GalNAc-Ts. Despite requiring transfection of cells with the engineered transferases and enzymes for biosynthesis of bioorthogonal substrates, the tactic complements methods in molecular biology. For complete details on the use and execution of this protocol, please refer to Schumann et al. (2020)¹, Cioce et al. (2021)², and Cioce et al. (2022)³.

SUBMITTER: Calle B

PROVIDER: S-EPMC9843269 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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Bump-and-hole engineering of human polypeptide N-acetylgalactosamine transferases to dissect their protein substrates and glycosylation sites in cells.

Calle Beatriz B Gonzalez-Rodriguez Edgar E Mahoney Keira E KE Cioce Anna A Bineva-Todd Ganka G Tastan Omur Y OY Roustan Chloe C Flynn Helen H Malaker Stacy A SA Schumann Benjamin B

STAR protocols 20230111 1

Despite the known disease relevance of glycans, the biological function and substrate specificities of individual glycosyltransferases are often ill-defined. Here, we describe a protocol to develop chemical, bioorthogonal reporters for the activity of the GalNAc-T family of glycosyltransferases using a tactic termed bump-and-hole engineering. This allows identification of the protein substrates and glycosylation sites of single GalNAc-Ts. Despite requiring transfection of cells with the engineer ...[more]

PMID: 36633947

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Project description:O-Glycan biosynthesis is initiated by the transfer of N-acetylgalactosamine (GalNAc) from a nucleotide sugar donor (UDP-GalNAc) to Ser/Thr residues of an acceptor substrate. The detailed transfer mechanism, catalyzed by the UDP-GalNAc polypeptide:N-acetyl-alpha-galactosaminyltransferases (ppGalNAcTs), remains unclear despite structural information available for several isoforms in complex with substrates at various stages along the catalytic pathway. We used all-atom molecular dynamics simulations with explicit solvent and counterions to study the conformational dynamics of ppGalNAcT-2 in several enzymatic states along the catalytic pathway. ppGalNAcT-2 is simulated both in the presence and in the absence of substrates and reaction products to examine the role of conformational changes in ligand binding. In multiple 40-ns-long simulations of more than 600 ns total run time, we studied systems ranging from 45,000 to 95,000 atoms. Our simulations accurately identified dynamically active regions of the protein, as previously revealed by the X-ray structures, and permitted a detailed, atomistic description of the conformational changes of loops near the active site and the characterization of the ensemble of structures adopted by the transferase complex on the transition pathway between the ligand-bound and ligand-free states. In particular, the conformational transition of a functional loop adjacent to the active site from closed (active) to open (inactive) is correlated with the rotameric state of the conserved residue W331. Analysis of water dynamics in the active site revealed that internal water molecules have an important role in enhancing the enzyme flexibility. We also found evidence that charged side chains in the active site rearrange during site opening to facilitate ligand binding. Our results are consistent with the single-displacement transfer mechanism previously proposed for ppGalNAcTs based on X-ray structures and mutagenesis data and provide new evidence for possible functional roles of certain amino acids conserved across several isoforms.

Dataset Information

Bump-and-hole engineering of human polypeptide N-acetylgalactosamine transferases to dissect their protein substrates and glycosylation sites in cells.

Publications

Bump-and-hole engineering of human polypeptide N-acetylgalactosamine transferases to dissect their protein substrates and glycosylation sites in cells.

Similar Datasets

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