Interactions of smads and WW domains of proteins involved in TGF-ß signalling
Year: 2012
Type: dissertation
Abstract: Smad proteins are the mediators of the TGF-ß signalling pathway that controls cell growth and specific cell differentiations. Defects in this pathway can lead to rapid cell growth resulting in cancer diseases. In response to TGF-ß signalling, Smad2 and 3 are activated and transferred with the help of Smad4 to the nucleus, where they regulate the transcription of target genes. The inhibitor Smad7 can negatively regulate the TGF-ß pathway by triggering TGF ß receptor I degradation. Smad proteins interact with other proteins through their linker region, which connects the DNA binding domain named MH1 with a TGF-ß-receptor-binding domain known as MH2 domain. The Smad2/3 linker region consists of several (S/T)P motifs that can be phosphorylated by different kinases. The Smad7 linker region is not phosphorylated but contains as Smad2 and 3 a PPxY (PY) motif that is recognized by WW domains. WW domains are small binding domains present in around 170 proteins of variable function involved in many cellular processes including transcription, splicing or protein degradation among others. In the main part of this work the interactions of R-Smad3 with the proteins Nedd4L and Pin1 were studied to understand the role of the Smad3 linker phosphorylation and its influence on WW domain binding. In the second part the interactions of the inhibitor Smad7 and the proteins Nedd4L and TAZ were characterized to obtain more information about their role in TGF-ß inhibition. In both parts the aim was to understand how specificity can be achieved by the WW domains. The structures of six WW domain - Smad complexes were solved and similarities and differences were pointed out. At the start the interaction of Nedd4L and the phosphorylated Smad2/3 linker, which leads to Smad2/3 degradation, has been studied. Using isothermal titration calorimetry (ITC) and nuclear magnetic resonance spectroscopy (NMR) it could be shown that of all four Nedd4L WW domains the second domain binds to the phosphorylated Smad2/3 pT[PY] motif with the highest affinity. The NMR structure of the Nedd4L WW2 – Smad3 pT[PY] complex revealed that the PPPxY site of the ligand is bound with all proline residues in trans configuration and the tyrosine residue accommodated in the second loop of the WW domain. The novelty of the complex resides in the binding of the phosphate group, which is specifically recognized by amino acids K378 and R380 in the first loop region. This new interaction has been corroborated by Nedd4L WW2 point mutations and by 31P-NMR titration experiments. Furthermore, the binding of the phosphorylated Smad3 linker to the WW domain of the peptidyl-prolyl isomerase Pin1 has been studied. Pin1 catalyses the cis-trans isomerisation of prolines specifically preceded by phosphorylated serines or threonines. The KD values obtained by ITC binding studies showed that the Pin1 WW domain strongly interacts with the Smad3 pT179P motif while bindings to the bi-phosphorylated Smad3 pS204P-pS208P and pS208P-pS213P peptides are weaker or barely observable as in the case of the monophosphorylated Smad3 pS204P peptide. To understand these differences in the binding affinities the structure of the Pin1 WW - Smad3 pT179P complex and of the Pin1 WW - Smad3 pS208P-pS213P complex was solved by solution NMR. It could be shown that in both complexes only one p(S/T)P motif is bound, also in trans-configuration, by the Pin1 WW domain involving amino acids of the first ß-stand and that the affinity is enhanced through the presence of a negatively charged amino acid at position x-1 and of a proline at position x-2 of the p(S/T)P motif. The influence of the phosphate group on binding the Pin1 WW domain was pointed out by 31P titrations. When compared to the Nedd4L WW2 recognition of the Smad3 pT179[PY] motif the Pin1 WW - Smad3 pT179P complex reveals a different binding mode. On the one hand the phosphate group is bound at different locations within the domains on the other hand the peptide is bound from the N- to C-terminus by the Pin1 WW domain and in opposite direction from the C- to N-terminus by the Nedd4L WW2 domain. The different modes of Smad3 recognition may reflect the different outcomes of these two interactions. ITC measurements of Nedd4L WW2 and Smad3 pT179[PY] as well as of Pin1 WW and Smad3 pS208P-pS213P at different proton concentrations revealed that in both cases the binding affinity increases with the pH values. The highest affinity was observed at pH 8.0. This information was used to optimize the conditions for the NMR experiments, but could also play a role in the regulation of protein binding in the physiological environment of the cell. In order to understand the role of the other WW domains present in Nedd4L, Smad3 fragments including the PY motif and other pSP sites were titrated with the three possible Nedd4L WW pairs. Of the three consecutive pairs present in Nedd4L, only the WW2-WW3 pair showed high affinity for the extended Smad3 sequence. However, the determination of the structure of the Nedd4L WW2-WW3 module in solution bound to the Smad3 linker peptide pT[PY]-pS-pS (Smad3 176-211 segment tri-phosphorylated at T179, S204 and S208) posed a challenge owing to the complexity introduced by the poor NMR signal dispersion of the 80 amino acid region connecting WW2 and WW3 domains. To simplify the interpretation of the NMR data, the WW2-WW3 module was prepared as two separate fragments, which allowed the use of sequential isotope labelling. A disulfide-bond protein ligation strategy was applied to connect these two fragments. Using a stepwise assignment approach the complex structure with the Smad3 176-211 segment was studied in detail. In the second part of this work the question was raised how inhibitor Smad7 is recognized by the WW domain containing proteins Nedd4L and TAZ. Smad7 recruits Nedd4L for TGF-ß receptor I degradation in the cytoplasm while the interaction with TAZ mainly occurs in the nucleus. Through ITC binding studies of the individual Nedd4L WW domains and a 15 amino acid Smad7 linker peptide containing the PY motif it could be shown that the second WW domain is the preferred Smad7 binding domain. Solution NMR based structure determination of the Nedd4L WW2 domain bound to the Smad7[PY] peptide revealed that the high affinity is due to additional contacts of the C-terminal part of Smad7 and the ß1 sheet region of the Nedd4L WW2 domain and electrostatic interactions of the Smad7 N-terminus and the loop 1 region of the domain. Further it could be shown by ITC experiments that this affinity is not enhanced by the presence of additional Nedd4L WW domains. The co-factors YAP and its paralogue TAZ consist of two (YAP) and one (TAZ) WW domain(s) and have also been reported to interact with Smads. However, the interaction between YAP and Smad7 results in enhanced TGF-ß receptor degradation whereas binding of TAZ and Smad7 has not been studied yet. The sequence alignment of the WW domains of TAZ and YAP reveals a higher sequence identity between YAP WW1 and TAZ WW than between the YAP WW1 and WW2 domains. ITC experiments carried out in this thesis revealed that the first WW domain in YAP binds the 15 amino acid Smad7 peptide with a higher affinity than the second WW domain and equally well as TAZ WW. Structures of the YAP WW1 domain bound to PY motifs have been previously described but in this work the first structure for the WW domain of the human TAZ bound to a PY ligand was determined. The TAZ WW - Smad7[PY] complex is very similar to its Nedd4L WW2 counterpart in the PY motif-containing region and in the C-terminal extension, but differ in the way in which the Smad7 N-terminus is recognized. As a conclusion, the findings presented in this work expand the known structural and functional versatility of WW domains as protein-protein interaction modules. Further this work shows that the interaction of Nedd4L and Pin1 with Smad3 is regulated by linker phosphorylation up- and downstream of the PY motif, which is carried out by special kinases in a precise order. Nedd4L binds to the Smad3 pT[PY] motif and two additional phosphorylation sites within Smad3 linker. The Pin1 WW domain only requires the Smad3 pTP motif with a single phosphorylation site for binding, which probably acts as the anchoring place for the prolyl-isomerase protein. Thus, Pin1 WW may be able to participate in the regulation of Smad3 as an active player of the Smad3 and Nedd4L interaction. Further, Nedd4L achieves high specificity by binding Smad3 following a two-step cooperative mode that involves the two vicinal domains WW2 and WW3. This ability of WW domains to recognize both pT[PY] and pS-pS motifs was previously unknown. The interaction of Nedd4L and Smad7 results in inhibition of the TGF-ß pathway through TGF-ß receptor I degradation. In this work it could be demonstrated that the single Nedd4L WW2 domain is sufficient to interact with the Smad7[PY] motif with high affinity. The individual WW domain of the adaptor protein TAZ also interacts with the inhibitor Smad but slightly weaker. Both complexes are very similar in the region containing the PY motif and also the C-terminal extension but they differ in the recognition of the N-term site by the domain. These differences may reflect the distinct functional outcomes of the two interactions. TAZ was previously known as a TGF-ß signalling agonist but in this work it is shown that TAZ also can play an inhibitory role in TGF-ß signalling as reported for its paralogue YAP.
Author: Görner, Nina
Language: English
Topic: TGF-β signaling in diseases
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W1536923016