Abstract

It has long been understood that some proteins undergo conformational transitions en route to the Michaelis Complex to allow chemistry. Examination of crystal structures of glycosyltransferase enzymes in the GT-B structural class reveals that the presence of ligand in the active site triggers an open-to-closed conformation transition, necessary for their catalytic functions. Herein, we describe microsecond molecular dynamics simulations of two distantly related glycosyltransferases that are part of the GT-B structural superfamily, HepI and GtfA. Simulations were performed using the open and closed conformations of these unbound proteins, respectively, and we sought to identify the major dynamical modes and communication networks that interconnect the open and closed structures. We provide the first reported evidence within the scope of our simulation parameters that the interconversion between open and closed conformations is a hierarchical multistep process which can be a conserved feature of enzymes of the same structural superfamily. Each of these motions involves of a collection of smaller molecular reorientations distributed across both domains, highlighting the complexities of protein dynamic involved in the interconversion process. Additionally, dynamic cross-correlation analysis was employed to explore the potential effect of distal residues on the catalytic efficiency of HepI. Multiple distal nonionizable residues of the C-terminal domain exhibit motions anticorrelated to positively charged residues in the active site in the N-terminal domain involved in substrate binding. Mutations of these residues resulted in a reduction in negatively correlated motions and an altered enzymatic efficiency that is dominated by lower Km values with kcat effectively unchanged. The findings suggest that residues with opposing conformational motions involved in the opening and closing of the bidomain HepI protein can allosterically alter the population and conformation of the “closed” state, essential to the formation of the Michaelis complex. The stabilization effects of these mutations likely equally influence the energetics of both the ground state and the transition state of the catalytic reaction, leading to the unaltered kcat. Our study provides new insights into the role of conformational dynamics in glycosyltransferase’s function and new modality to modulate enzymatic efficiency.

Highlights

  • Glycosylation is a highly regulated, ubiquitous biochemical process catalyzed by glycosyltransferase (GT, E.C. 2.4.x.y) enzymes

  • This is the first ever all atom, microsecond molecular dynamics (MD) simulation of GT-B glycosytransferases, and the study suggests that Cα root-mean-square deviation (Cα RMSD) by itself is not a good test to determine structural stability at higher timeframes, which aligns with other work utilizing MD to simulate multidomain proteins connected by linker regions [49]

  • The simulations of these two distantly related, but structurally conserved glycosyltransferases have given us insight into how the structural scaffold itself, and not the primary amino acid sequences, control the protein dynamic modes

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Summary

Introduction

Glycosylation is a highly regulated, ubiquitous biochemical process catalyzed by glycosyltransferase (GT, E.C. 2.4.x.y) enzymes. Significant research endeavors have identified and characterized key residues and regions linked to the enzymatic cycle in a diverse range of GTs [2,3,4,5,6,7,8,9]. These investigations provided a wealth of data, making it possible to develop an atomistic description of the molecular mechanism of GTs. An understanding of the functional dynamics of this important class of enzymes is paramount for inhibitor discovery, chimeric protein design, enzymatic control and regulation, among other studies that offer potentially beneficial clinical and industrial applications

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