Disorder in a Target for the Smad2 Mad Homology 2 Domain and Its Implications for Binding and Specificity

Abstract

The Smad2 Mad homology 2 (MH2) domain binds to a diverse group of proteins which do not share a common sequence motif. We have used NMR to investigate the structure of one of these interacting proteins, the Smad binding domain (SBD) of Smad anchor for receptor activation (SARA). Our results indicate that the unbound SBD is highly disordered and forms no stable secondary or tertiary structures. Additionally we have used fluorescence binding studies to study the interaction between the MH2 domain and SBD and find that no region of the SBD dominates the interaction between the MH2 and the SBD. Our results are consistent with a series of hydrophobic patches on the MH2 that are able to recognize disordered regions of proteins. These findings elucidate a mechanism by which a single domain (MH2) can specifically recognize a diverse set of proteins which are unrelated by sequence, lead to a clearer picture of how MH2 domains function in the transforming growth factor-␤-signaling pathway and suggest possible mechanisms for controlling interactions with MH2 domains.The TGF-␤ 1 superfamily of cytokines plays an essential role in a variety of cellular responses including differentiation, cell fate specification, and growth inhibition (1-4). Dysregulation of TGF-␤ signaling has been associated with many diseases, such as human cancers, fibrosis, hereditary hemorrhagic telangiectasia (2, 5), and Marfan syndrome (6). TGF-␤ signal transduction is mediated intracellularly by the Smad proteins, including the R-Smads (receptor-regulated Smads), the I-Smads (inhibitor Smads), and the Co-Smad (common Smad). Ligand binding to the TGF-␤ receptor complex or a homologous complex leads to receptor phosphorylation of the R-Smads (7, 8). Subsequently, the phosphorylated R-Smads can interact with the Co-Smad, Smad4, and accumulate in the nucleus (9, 10). In the nucleus the Smad proteins bind to transcription factors and promoter regions and play a role in the transcription of various genes (11, 12).The R-Smads and Smad4 consist of a conserved N-terminal Mad homology 1 (MH1) domain and a conserved C-terminal MH2 domain joined by a poorly conserved linker region. The MH1 domain is known to function in binding DNA (13). The MH2 domain appears to exhibit many different roles and binds to many different proteins. Although many modular protein domains involved in signal transduction, such as SH3, SH2, WW, PTB, and PDZ domains, interact with a recognizable sequence motif (14), MH2 domains appear to be able to bind to a wide variety of ligands that do not share a single common motif. For example the MH2 domain of Smad2 binds a plethora of non-homologous proteins including SARA (15), the TGF-␤ receptors, FoxH1 (16), Mixer (17), TGIF (18,19), CBP (20), AML1 (21,22), Ski (23,24), and SIP1 (25). These interacting partners represent a diverse group of protein types, encompassing receptors, membrane anchoring proteins, and transcription factors. Furthermore, they have no region of sequence or structural similarity in common. A key question is how the M...

Developmental expression of myostatin in cardiomyocytes and its effect on foetal and neonatal rat cardiomyocyte proliferation

“…The complex then translocates into the nucleus to bind promoter regions of target genes or interact with various transcription factors to regulate gene expression [33][34][35]. SMAD2 proteins are able to interact with a diverse range of proteins that do not share a common motif [35,36]. Hence the phenotypic outcome following SMAD activation is therefore likely to depend on the SMAD-binding transcription factors present within the nucleus at the time of their activation.…”

Selective inhibition of TGF-β responsive genes by Smad-interacting peptide aptamers from FoxH1, Lef1 and CBP

“…Mutational analysis indicates that the FoxH1 SIM binds to the same portion of the hydrophobic shallow groove used to bind the rigid coil motif in SARA, a portion of the hydrophobic groove that is not involved in the Smad-Smad-binding interface (Randall et al, 2004). The structural basis for Smad binding to CBP and Lef1 is not currently understood, although binding of Flag-CBP to HASmad3 was inhibited by coexpression of Ski protein, suggesting overlap in the binding sites of Ski and CBP (Wu et al, 2002) A recent report suggests that the shallow hydrophobic groove on Smad2 might bind a diverse group of unstructured protein motifs with little or no primary sequence similarity (Chong et al, 2004). The hydrophobic shallow groove has also been implicated in Smad-binding to the nuclear transport proteins involved in shuttling Smad protein from the cytoplasm into the nucleus Xu et al, 2002).…”

Coupling of tandem Smad ubiquitination regulatory factor (Smurf) WW domains modulates target specificity

“…Fluorescence binding experiments were performed at 20°C in buffer containing 40 mM Na 2 HPO 4 , pH 7.2, and 20 mM NaCl and 1 mM fresh β-mercaptoethanol. Fluorescence levels were monitored as peptide was added to the WW23 protein samples and data was fit as previously published (25). See SI Methods for more details.…”