Abstract
α-Mannoside β-1,6-N-acetylglucosaminyltransferase V (MGAT5) is a mammalian glycosyltransferase involved in complex N-glycan formation, which strongly drives cancer when overexpressed. Despite intense interest, the catalytic mechanism of MGAT5 is not known in detail, precluding therapeutic exploitation. We solved structures of MGAT5 complexed to glycosyl donor and acceptor ligands, revealing an unforeseen role for donor-induced loop rearrangements in controlling acceptor substrate engagement. QM/MM metadynamics simulations of MGAT5 catalysis highlight the key assisting role of Glu297 and reveal considerable conformational distortions imposed upon the glycosyl donor during transfer. Detailed mechanistic characterization of MGAT5 will aid inhibitor development to correct cancer-associated N-glycosylation.
Highlights
ABSTRACT: α-Mannoside β-1,6-N-acetylglucosaminyltransferase V (MGAT5) is a mammalian glycosyltransferase involved in complex N-glycan formation, which strongly drives cancer when overexpressed
The resulting branched GlcNAc-β-1,6-Man linkage is a precursor for the formation of complex tri- and tetra-antennary N-glycans, which are elaborated in the trans-Golgi by the addition of Gal-β1,4-GlcNAc (LacNAc) disaccharides and sialic acids.[8]
The β-1,6 linked products of MGAT5 activity are preferentially elaborated by poly-LacNAc repeats (Figure 1),[13] which bind galectins with higher avidity and affinity
Summary
ABSTRACT: α-Mannoside β-1,6-N-acetylglucosaminyltransferase V (MGAT5) is a mammalian glycosyltransferase involved in complex N-glycan formation, which strongly drives cancer when overexpressed. M592 was substantially displaced from its binary complex position by the structured Lys279−Gly[293] loop (Figure 4b; Figure S7a), enabling a water mediated H-bonding network to form between UDP, Glu[297], and the acceptor α-mannose This H-bonding network involves a direct interaction between Glu[297] and the α-mannose O6, creating a catalytically poised active site in which acceptor nucleophile deprotonation is possible. MM simulations based upon our structural data provide a detailed description of MGAT5 catalyzed transfer, confirming the essential assisting role of Glu[297] in the reaction mechanism and capturing the conformational itinerary undertaken by GlcNAc throughout its migration from donor to acceptor. Michaelis−Menten kinetics graphs, MGAT5 ribbon and surface representations, root-mean-square deviations between different MGAT5 crystal forms, STARANISO2 calculated plots of reciprocal lattice points, comparison of M592 complex with previously reported MGAT5 acceptor complex, UDP-2FGlc ternary complex, MGAT5 acceptor subsite surfaces, analysis of protein atom root mean square displacements during the MD simulation timecourse, distribution of Michaelis complex C1−O6 distances during the MD simulation, data collection and refinement statistics, PDB accession codes, cloning primers, methods, and supporting references (PDF)
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