Substrate-induced Conformational Changes and Dynamics of UDP-N-Acetylgalactosamine:Polypeptide N-Acetylgalactosaminyltransferase-2

Abstract

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.

A computational and experimental study of O-glycosylation. Catalysis by human UDP-GalNAc polypeptide:GalNAc transferase-T2

“…Consequently, a double displacement seems unlikely or would require a large conformational change. The latter is not observed on the timescale of the MD simulations performed on the Michaelis complex of human GalNAc-T2 by Milac et al 33 On the other hand, their results are more consistent with a front-side mechanism, since the distance between the glycosidic oxygen and the nucleophilic hydroxyl group is about 3 Å and is maintained nearly constant during the simulation, which would be at least structurally consistent with a nucleophilic role of the acceptor. 33 These results, together with the available X-ray structures and site-directed mutagenesis data, point to a single-displacement mechanism as the most likely one.…”

QM/MM Studies Reveal How Substrate–Substrate and Enzyme–Substrate Interactions Modulate Retaining Glycosyltransferases Catalysis and Mechanism

“…Crystallographic structures of GalNAc-T2 show that the only possible nucleophilic residues that could act in a double-displacement mechanism are positioned $7 Å away from C1, suggesting that this is not the reaction mechanism followed by the enzyme (Fritz, Hurley, Trinh, Shiloach, & Tabak, 2004;Fritz, Raman, & Tabak, 2006;Kubota et al, 2006;Lira-Navarrete et al, 2014). Long timescale MD simulations performed by Milac et al did not change this observation (Milac, Buchete, Fritz, Hummer, & Tabak, 2007). Their simulations are more consistent with a front-side attack, as the acceptor hydroxyl (OA) is found at $3 Å all along the simulations.…”

UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase- 6 (pp-GalNAc-T6): Role in Cancer and Prospects as a Drug Target

“…There has been two mechanisms proposed [19,30]. The first is a double replacement mechanism, in which the side chain amide NH2 from Asn-383 (Gln-346 in pp-GalNAc-T10) acts as a nucleophile and attacks the anomeric carbon of the GalNAc ring, and this leads to the formation of an enzyme-glycosyl intermediate [19].…”

“…The other proposed mechanism is a front-side attack mechanism [30]. This was postulated as an alternative mechanism because of doubt about the ability of asparagine and glutamine to act as nucleophiles as well as the potential nucleophile's distance from the β-phosphate oxygen (approximately 7 Å In pp-GalNAc-T2) [30].…”

“…This was postulated as an alternative mechanism because of doubt about the ability of asparagine and glutamine to act as nucleophiles as well as the potential nucleophile's distance from the β-phosphate oxygen (approximately 7 Å In pp-GalNAc-T2) [30]. In the front side attack mechanism the hydroxyl group of the protein substrate acts as the nucleophile attacking the anomeric carbon.…”