Structural characterization by NMR of the natively unfolded extracellular domain of β-dystroglycan:: Toward the identification of the binding epitope for α-dystroglycan

被引:15
作者
Bozzi, M
Bianchi, M
Sciandra, F
Paci, M
Giardina, B
Brancaccio, A
Cicero, DO
机构
[1] Univ Cattolica Sacro Cuore, Ist Chim Riconoscimento Mol, Ist Biochim & Biochim Clin, CNR, I-00168 Rome, Italy
[2] Univ Roma Tor Vergata, Dipartimento Sci & Tecnol Chim, I-00133 Rome, Italy
[3] INFM, Sez B, Rome, Italy
关键词
D O I
10.1021/bi034867w
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 (C-13,N-15) form and studied by multidimensional NMR. Although it represents a natively unfolded protein domain, we obtained an almost complete backbone assignment. Chemical shift index, H-1-N-15 heteronuclear single-quantum coherence and nuclear Overhauser effect (HSQC-NOESY) spectra and (3)J(HN,Halpha) coupling constant values confirm that this protein is highly disordered, but H-1-N-15 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.
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页码:13717 / 13724
页数:8
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