The Structure of the T190M Mutant of Murine α-Dystroglycan at High Resolution: Insight into the Molecular Basis of a Primary Dystroglycanopathy.
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.
Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Roma 00168, Italy.
Manuela
M
Bozzi
Istituto di Cristallografia-CNR, Trieste Outstation, Trieste, 34149, Italy.
Alberto
A
Cassetta
Istituto di Cristallografia-CNR, Trieste Outstation, Trieste, 34149, Italy.
Sonia
S
Covaceuszach
School of Biochemistry, University of Bristol, Bristol, BS8 1TD, United Kingdom.
Maria Giulia
MG
Bigotti
Department of Physics, University of Bielefeld, 33615, Bielefeld, Germany.
Saskia
S
Bannister
Department of Physics, University of Bielefeld, 33615, Bielefeld, Germany.
Wolfgang
W
Hübner
Istituto di Chimica del Riconoscimento Molecolare-CNR, c/o Università Cattolica del Sacro Cuore, Roma, 00168, Italy.
Francesca
F
Sciandra
Istituto di Cristallografia-CNR, Trieste Outstation, Trieste, 34149, Italy.
Doriano
D
Lamba
School of Biochemistry, University of Bristol, Bristol, BS8 1TD, United Kingdom; Istituto di Chimica del Riconoscimento Molecolare-CNR, c/o Università Cattolica del Sacro Cuore, Roma, 00168, Italy.
Andrea
A
Brancaccio
D000818:Animals; D045744:Cell Line, Tumor; D049030:Dystroglycans / Q000737:chemistry*; D006801:Humans; D006860:Hydrogen Bonding; D051379:Mice; D008853:Microscopy; D008958:Models, Molecular; D049288:Muscular Dystrophies, Limb-Girdle / Q000378:metabolism*; D050505:Mutant Proteins / Q000737:chemistry*; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D014961:X-Ray Diffraction
D050505:Mutant Proteins; D049030:Dystroglycans