Identification and Modeling of a GT-A Fold in the α-Dystroglycan Glycosylating Enzyme LARGE1

Abstract

The acetylglucosaminyltransferase-like protein LARGE1 is an enzyme that is responsible for the final steps of the post-translational modifications of dystroglycan (DG), a membrane receptor that links the cytoskeleton with the extracellular matrix in the skeletal muscle and in a variety of other tissues. LARGE1 acts by adding the repeating disaccharide unit [-3Xyl-α1,3GlcAβ1-] to the extracellular portion of the DG complex (α-DG); defects in the LARGE1 gene result in an aberrant glycosylation of α-DG and consequent impairment of its binding to laminin, eventually affecting the connection between the cell and the extracellular environment. In the skeletal muscle, this leads to degeneration of the muscular tissue and muscular dystrophy. So far, a few missense mutations have been identified within the LARGE1 protein and linked to congenital muscular dystrophy, and because no structural information is available on this enzyme, our understanding of the molecular mechanisms underlying these pathologies is still very limited. Here, we generated a 3D model structure of the two catalytic domains of LARGE1, combining different molecular modeling approaches. Furthermore, by using molecular dynamics simulations, we analyzed the effect on the structure and stability of the first catalytic domain of the pathological missense mutation S331F that gives rise to a severe form of muscle–eye–brain disease.

High degree of conservation of the enzymes synthesizing the laminin-binding glycoepitope of α-dystroglycan

“…Upon the addition of the RboP dimer and prior to the synthesis of the matriglycan polymer, a dimer of xylole and glucuronic acid is added by RXYLT1, a ribitol xylosyltransferase 1 (ribitol β 1,4-Xylosyltransferase) [ 63 ] and B4GAT1 (β-1,4-glucuronyltransferase 1) formerly known as B3GNT1 [ 64 , 65 ]. Then, LARGE1 adds the repeating disaccharide unit [-3Xyl-α1,3GlcA β 1-] n [ 66 ] by employing its double capacity as a xylosyltransferase (domain 1) and a glucuronyltransferase (domain 2) [ 67 ]: the tandemly repeated polymer of these disaccharide units thus generated is called matriglycan, [ 20 ]. The N-terminal domain of α-DG binds LARGE and it could act as a chaperone for directing the enzymatic activity of LARGE towards its own mucin-like domain [ 68 , 69 ].…”

Conserved Conformational Hierarchy across Functionally Divergent Glycosyltransferases of the GT-B Structural Superfamily as Determined from Microsecond Molecular Dynamics

“…The majority of the simulations to date were performed using protein structures built from homology models as their structural starting point due to the lack of available crystal structures for the desired proteins. Several GT-As, including GT MG517 from Mycoplasma genitalium [ 23 ], and LARGE1 from humans [ 24 ], together with two GT-Bs, GnT-V from humans [ 25 ] and HepIII from Klebsiella pneumonia [ 26 ], have been modeled and short molecular dynamics simulations have been performed to refine the structure and to corroborate or provide new insight for substrate/protein interactions. For those GTs that have crystal structures available, short (<300 ns) all atom or longer (10 µs) course grain molecular dynamic simulations have been performed.…”

Ligand Induced Conformational and Dynamical Changes in a GT-B Glycosyltransferase: Molecular Dynamic Simulations of Heptosyltransferase I Apo, Binary and Ternary Complexes

“…[26,27] In addition, simulations have been used in tandem with homology modeling to further study GTs when crystal structures are unavailable. [28][29][30][31] Heptosyltransferase I (HepI) is Glycosyltransferase in the GT9 family with a characteristic GT-B fold. It consists of two domains with β/α/β Rossman-like folds connected by a linker (Figure 1A).…”

“…[26, 27] In addition, simulations have been used in tandem with homology modeling to further study GTs when crystal structures are unavailable. [28–31]…”