The Structure of the T190M Mutant of Murine α-Dystroglycan at High Resolution: Insight into the Molecular Basis of a Primary Dystroglycanopathy

Abstract

The severe dystroglycanopathy known as a form of limb-girdle muscular dystrophy (LGMD2P) is an autosomal recessive disease caused by the point mutation T192M in α-dystroglycan. Functional expression analysis in vitro and in vivo indicated that the mutation was responsible for a decrease in posttranslational glycosylation of dystroglycan, eventually interfering with its extracellular-matrix receptor function and laminin binding in skeletal muscle and brain. The X-ray crystal structure of the missense variant T190M of the murine N-terminal domain of α-dystroglycan (50-313) has been determined, and showed an overall topology (Ig-like domain followed by a basket-shaped domain reminiscent of the small subunit ribosomal protein S6) very similar to that of the wild-type structure. The crystallographic analysis revealed a change of the conformation assumed by the highly flexible loop encompassing residues 159–180. Moreover, a solvent shell reorganization around Met190 affects the interaction between the B1–B5 anti-parallel strands forming part of the floor of the basket-shaped domain, with likely repercussions on the folding stability of the protein domain(s) and on the overall molecular flexibility. Chemical denaturation and limited proteolysis experiments point to a decreased stability of the T190M variant with respect to its wild-type counterpart. This mutation may render the entire L-shaped protein architecture less flexible. The overall reduced flexibility and stability may affect the functional properties of α-dystroglycan via negatively influencing its binding behavior to factors needed for dystroglycan maturation, and may lay the molecular basis of the T190M-driven primary dystroglycanopathy.

Induced Structural Disorder as a Molecular Mechanism for Enzyme Dysfunction in Phosphoglucomutase 1 Deficiency

“…In contrast, other missense mutants, such as D263Y and T19A, show little change from WT enzyme in this assay, suggesting that their defects discretely affect catalysis/ligand binding, rather than protein structure. As many other proteins have missense variants that show differences in such assays (e.g., [34–36]), it is possible that induced disorder is a common outcome of disease-related mutations, which is underappreciated due to lack of structural information.…”

An evaluation of the evolution of the gene structure of dystroglycan

“…3b, d). However, the intron location does not correspond with the protein domain structure or the IG1 domain boundaries; the IG1-intron interrupts the sequence encoding the middle of the third β-strand of the IG1 domain [11, 14] (Fig. 3e).…”

“…Indeed, the N-terminal region in isolation displays a residual laminin-binding activity [11] and is likely to be important for directing the actions of a plethora of enzymes required for the glycosylation of α-DG [7, 14]. Based on pioneering recombinant protein analysis, the N-terminal domain of α-DG has been suggested to represent an autonomous module [15].…”

“…This module can be liberated by furin-driven proteolysis [16, 17] within the extracellular space and/or body fluids [18–21]. It is also speculated that the IG1 domain might function in self-recognition (in cis ) of carbohydrate moieties that protrude from the neighbouring mucin-like region, and therefore could have additional functions within the glycosylation and maturation pathway of the dystroglycan precursor molecule [14]. …”

A molecular overview of the primary dystroglycanopathies

“…29 Due to the observed high flexibility within the two subdomains of α-DG N-terminal region, itcan be speculated that some of these mutations might also affect the interactions between DG and other enzymes important for the post-translational glycosylation of DG in the Golgi (decoration process) still without inhibiting the maturation of the α/β complex into its two subunits 30,31. DAG1 mutations are rare, recessive mutations that are found in consanguineous families.…”