Structural analysis of the Smad2-MAN1 interaction that regulates transforming growth factor-β signaling at the inner nuclear membrane.
MAN1, an integral protein of the inner nuclear membrane, influences transforming growth factor-β (TGF-β) signaling by directly interacting with R-Smads. Heterozygous loss of function mutations in the gene encoding MAN1 cause sclerosing bone dysplasias and an increased level of TGF-β signaling in cells. As a first step in elucidating the mechanism of TGF-β pathway regulation by MAN1, we characterized the structure of the MAN1 C-terminal region that binds Smad2. Using nuclear magnetic resonance spectroscopy, we observed that this region is comprised of a winged helix domain, a structurally heterogeneous linker, a U2AF homology motif (UHM) domain, and a disordered C-terminus. From nuclear magnetic resonance and small-angle X-ray scattering data, we calculated a family of models for this MAN1 region. Our data indicate that the linker plays the role of an intramolecular UHM ligand motif (ULM) interacting with the UHM domain. We mapped the Smad2 binding site onto the MAN1 structure by combining GST pull-down, fluorescence, and yeast two-hybrid approaches. The linker region, the UHM domain, and the C-terminus are necessary for Smad2 binding with a micromolar affinity. Moreover, the intramolecular interaction between the linker and the UHM domain is critical for Smad2 binding. On the basis of the structural heterogeneity and binding properties of the linker, we suggest that it can interact with other UHM domains, thus regulating the MAN1-Smad2 interaction.
Kondé
Emilie
E
Laboratoire de Biologie Structurale et Radiobiologie, URA CNRS 2096, CEA Saclay, 91190 Gif-sur-Yvette, France.
Bourgeois
Benjamin
B
Tellier-Lebegue
Carine
C
Wu
Wei
W
Pérez
Javier
J
Caputo
Sandrine
S
Attanda
Wika
W
Gasparini
Sylvaine
S
Charbonnier
Jean-Baptiste
JB
Gilquin
Bernard
B
Worman
Howard J
HJ
Zinn-Justin
Sophie
S
D020816:Amino Acid Motifs / Q000235:genetics; D006801:Humans; D009154:Mutation; D009685:Nuclear Envelope / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D051899:Smad2 Protein; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*; D014965:X-Rays
C497782:SMAD2 protein, human; D051899:Smad2 Protein; D016212:Transforming Growth Factor beta