NMR characterization of intramolecular interaction of osteopontin, an intrinsically disordered protein with cryptic integrin-binding motifs.
Osteopontin (OPN) is an integrin-binding protein found in a variety of tissues and physiological fluids and is involved in divergent biological processes such as migration, adhesion and signaling in integrin-independent as well as dependent manners. The adhesive activity of this protein is modulated upon cleavage by thrombin at the central part of the molecule, in the vicinity of the integrin-binding sequences. Although detailed structural characterization is crucial for further understanding of the regulatory mechanisms of the OPN functions, its intrinsically disordered property hampers in-depth conformational analyses. Here we report an NMR study of mouse OPN and its N-terminal thrombin-cleavage product to characterize intramolecular interaction of this molecule. Paramagnetic relaxation enhancement experiment revealed that OPN exhibits a long-range intramolecular interaction between the N- and C-terminal regions. Furthermore, our NMR data showed that anti-OPN antibody OPN1.2, whose reactivity is impaired by deletion or amino acid substitutions of the arginine-aspartate-glycine integrin-binding motif, binds the N-terminal side of the integrin-binding motifs suggesting the existence of intramolecular interaction. These data suggest that functional interactions of OPN with integrins and the other binding partners can be modulated by the intramolecular interactions.
Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan.
Yoshiki
Y
Yamaguchi
Shinya
S
Hanashima
Hirokazu
H
Yagi
Yutaka
Y
Takahashi
Hiroaki
H
Sasakawa
Eiji
E
Kurimoto
Takeshi
T
Iguchi
Shigeyuki
S
Kon
Toshimitsu
T
Uede
Koichi
K
Kato
D020816:Amino Acid Motifs; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites; D002942:Circular Dichroism; D018604:Epitope Mapping; D016023:Integrins / Q000378:metabolism*; D051379:Mice; D019906:Nuclear Magnetic Resonance, Biomolecular; D053495:Osteopontin / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D016023:Integrins; D011994:Recombinant Proteins; D053495:Osteopontin