title: Structural characterization by NMR of the natively unfolded extracellular domain of beta-dystroglycan: toward the identification of the binding epitope for alpha-dystroglycan.
abstract: 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: alpha-DG, membrane-associated and highly glycosylated, and the transmembrane beta-DG. Recently, by solid-phase binding assays and NMR experiments, we have shown that the C-terminal domain of alpha-DG interacts with a recombinant extracellular fragment of beta-DG (positions 654-750) independently from glycosylation and that the linear binding epitope is located between residues 550 and 565 of alpha-DG. In order to elucidate which moieties of beta-DG are specifically involved in the complex with alpha-DG, the ectodomain has been recombinantly expressed and purified in a labeled ((13)C,(15)N) form and studied by multidimensional NMR. Although it represents a natively unfolded protein domain, we obtained an almost complete backbone assignment. Chemical shift index, (1)H-(15)N heteronuclear single-quantum coherence and nuclear Overhauser effect (HSQC-NOESY) spectra and (3)J(HN,H)(alpha) coupling constant values confirm that this protein is highly disordered, but (1)H-(15)N 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 beta-DG(654-750) to the C-terminal region of the alpha subunit, alpha-DG(485-620), has been investigated, showing that the region of beta-DG(654-750) between residues 691 and 719 is involved in the interaction.
authors: Istituto di Chimica del Riconoscimento Molecolare c/o Istituto di Biochimica e Biochimica Clinica, Consiglio Nazionale della Ricerche, Università Cattolica del Sacro Cuore, Largo Francesco Vito 1, 00168 Rome, Italy.
authors: Manuela
authors: M
authors: Bozzi
authors: Marzia
authors: M
authors: Bianchi
authors: Francesca
authors: F
authors: Sciandra
authors: Maurizio
authors: M
authors: Paci
authors: Bruno
authors: B
authors: Giardina
authors: Andrea
authors: A
authors: Brancaccio
authors: Daniel O
authors: DO
authors: Cicero
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D049030:Dystroglycans; D000939:Epitopes / Q000032:analysis*; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D003598:Cytoskeletal Proteins; D000939:Epitopes; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D049030:Dystroglycans