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

MshA is a GT-B glycosyltransferase
catalyzing the first step in
the biosynthesis of mycothiol. While many GT-B enzymes undergo an
open-to-closed transition, MshA is unique because its 97° rotation
is beyond the usual range of 10–25°. Molecular dynamics
(MD) simulations were carried out for MshA in both ligand bound and
unbound states to investigate the effect of ligand binding on localized
protein dynamics and its conformational free energy landscape. Simulations
showed that both the unliganded “opened” and liganded
“closed” forms of the enzyme sample a wide degree of
dihedral angles and interdomain distances with relatively low overlapping
populations. Calculation of the free energy surface using replica
exchange MD for the apo “opened” and an artificial generated
apo “closed” structure revealed overlaps in the geometries
sampled, allowing calculation of a barrier of 2 kcal/mol for the open-to-closed
transition in the absence of ligands. MD simulations of fully liganded
MshA revealed a smaller sampling of the dihedral angles. The localized
protein fluctuation changes suggest that UDP-GlcNAc binding activates
the motions of loops in the 1-l-myo-inositol-1-phosphate
(I1P)-binding site despite little change in the interactions with
UDP-GlcNAc. Circular dichroism, intrinsic fluorescence spectroscopy,
and mutagenesis studies were used to confirm the ligand-induced structural
changes in MshA. The results support a proposed mechanism where UDP-GlcNAc
binds with rigid interactions to the C-terminal domain of MshA and
activates flexible loops in the N-terminal domain for binding and
positioning of I1P. This model can be used for future structure-based
drug development of inhibitors of the mycothiol biosynthetic pathway.