HDX-MS Reveals Substrate-Dependent, Localized EX1 Conformational Dynamics in the Retaining GT-B Glycosyltransferase, MshA

Abstract

Glycosyltransferases (GTs) are well-characterized with respect to static 3D structures and molecular dynamics simulations, but there is a lack of reports on in-solution dynamics on time scales relevant to turnover. Here, backbone amide hydrogen/deuterium exchange followed by mass spectrometry (HDX-MS) was used to investigate the in-solution dynamics of the model retaining GT MshA from Corynebacterium glutamicum (CgMshA). CgMshA has a GT-B fold and catalyzes the transfer of N-acetyl-glucosamine (GlcNAc) from UDP−GlcNAc to L-myoinositol-1-phosphate in the first step in mycothiol biosynthesis. HDX-MS results identify several key regions of conformational changes in response to UDP−GlcNAc binding, including residues 159−198 in the N-terminal domain and residues 323−354 in the C-terminal domain. These regions also exhibited substratedependent EX1 exchange kinetics consistent with conformational tension on the milliseconds to seconds time scale. A potential source of this conformational change is the flexible β4/α5 loop in the C-terminal domain, which sits at the interface of the two domains and likely interacts with the GlcNAc ring of UDP−GlcNAc. In contrast to UDP−GlcNAc, the UDP−CgMshA product complex exhibited severe decreases in deuterium incorporation, suggesting a less dynamic conformation. The HDX-MS results are complemented by solvent viscosity effects of 1.8−2.3 on the CgMshA k cat value, which are consistent with product release as a ratedetermining step and possibly a direct role for protein dynamics in catalysis. The identification of in-solution dynamics that are sensitive to substrate binding allows for the proposal of a more detailed mechanism in CgMshA including conformation tension between the donor sugar and the flexible C-terminal domain β4/α5 loop providing sufficient conformational sampling for substrateassisted catalysis to occur.

Specificity determinants revealed by the structure of glycosyltransferase Campylobacter concisusPglA

“…The N-acetyl sugar offers no stabilization of either enzyme (data not shown) emphasizing the affinity afforded by the UDP moiety. The results are consistent with known specificity of C. consisus PglA enzymes for UDP-GalNAc and with previous observations that the resulting UDP binds and stabilizes the GT-B fold (Karki et al, 2023).…”

In Vitro and In Silico Explorations of the Protein Conformational Changes of Corynebacterium glutamicum MshA, a Model Retaining GT-B Glycosyltransferase

“…The UDP product and UDP-GlcNAc substrate MshA complexes behave more similarly to one another with similar denaturation curves and decreases in intrinsic fluorescence. These results are in line with recent HDX-MS data showing that binding UDP or UDP-GlcNAc affects deuterium incorporation into similar regions of MshA …”

“…This view obscures a role for changes in localized protein dynamics in response to ligand binding and in shifting equilibrium from the open to closed state. For example, a recent HDX-MS study on MshA identified several regions that exhibit changes in deuterium uptake upon formation of liganded MshA complexes at areas distal to the site of ligand binding . In this study, we aim to utilize molecular dynamics (MD) and replica exchange molecular dynamics (REMD) simulations to identify the regions of MshA that undergo changes in fluctuations with respect to open/closed conformations and ligand binding over the 2 μs simulation.…”

Cell-free expression and biochemical characterization of polysaccharide-synthesizing glycosyltransferases

“…The glycosylation reaction in these GTs can proceed with either inversion or retention of stereochemistry at the C1 position of the nucleotide sugar. Some of these GTs combine both polymerizations of nucleotide sugars in an S N 2-like nucleophilic displacement reaction and translocate the growing polymer across the plasma membrane through its own transmembrane pore 6, 7 . The glycan polymers synthesized by these GTs contain tens of thousands of monomeric sugar units and can be extremely long 4, 8 .…”