Structural characterization by NMR of the natively unfolded extracellular domain of b-dystroglycan: towards the identification of the binding epitope for a-dystroglycan

Dystroglycan (DG) is an adhesion molecule playing a crucial role for tissue stability during both early embriogenesis and adulthood and is composed by two tightly interacting subunits:  α-DG, membrane-associated and highly glycosylated, and the transmembrane β-DG. Recently, by solid-phase binding assays and NMR experiments, we have shown that the C-terminal domain of α-DG interacts with a recombinant extracellular fragment of β-DG (positions 654−750) independently from glycosylation and that the linear binding epitope is located between residues 550 and 565 of α-DG. In order to elucidate which moieties of β-DG are specifically involved in the complex with α-DG, the ectodomain has been recombinantly expressed and purified in a labeled (13C,15N) form and studied by multidimensional NMR. Although it represents a natively unfolded protein domain, we obtained an almost complete backbone assignment. Chemical shift index, 1H−15N heteronuclear single-quantum coherence and nuclear Overhauser effect (HSQC−NOESY) spectra and 3JHN,Hα coupling constant values confirm that this protein is highly disordered, but 1H−15N steady-state NOE experiments indicate that the protein presents two regions of different mobility. The first one, between residues 659 and 722, is characterized by a limited degree of mobility, whereas the C-terminal portion, containing about 30 amino acids, is highly flexible. The binding of β-DG(654−750) to the C-terminal region of the α subunit, α-DG(485−620), has been investigated, showing that the region of β-DG(654−750) between residues 691 and 719 is involved in the interaction.