Volume 22, Issue 6, Pages 731-740 (June 2006) Structure of the Tfb1/p53 Complex: Insights into the Interaction between the p62/Tfb1 Subunit of TFIIH and the Activation Domain of p53  Paola Di Lello, Lisa M. Miller Jenkins, Tamara N. Jones, Bao D. Nguyen, Toshiaki Hara, Hiroshi Yamaguchi, Jimmy D. Dikeakos, Ettore Appella, Pascale Legault, James G. Omichinski  Molecular Cell  Volume 22, Issue 6, Pages 731-740 (June 2006) DOI: 10.1016/j.molcel.2006.05.007 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 In Vitro Binding Studies of Tfb1 and p62 with the TAD of p53 (A) Tfb1 and p62 bind the TAD of p53, but not to the TAD1 subdomain. (Upper panel) GST-Tfb11–115 (1 μM) was coupled to GSH-Sepharose and incubated with 1.0 μM, 0.5 μM, and 0.1 μM of p531–73 (lanes 5–7) or with 1.0 μM, 0.5 μM, and 0.1 μM of p531–40 (lanes 8–10). (Lower panel) GST-p621–127 (1 μM) was coupled to GSH-Sepharose and incubated with 1.0 μM, 0.5 μM, and 0.1 μM p531–73 (lanes 5–7) or 1.0 μM, 0.5 μM, and 0.1 μM of p531–40 (lanes 8–10). In both experiments, neither 1 μM p531–73 (lane 3) nor 1 μM p531–40 (lane 4) was retained by GST (1 μM) coupled to GSH-Sepharose. Purified p531–73 (lane 1) and p531–40 (lane 2) were loaded as controls. The p53 peptides were detected with anti-p53 antibody. Coomassie staining verified equivalent inputs of GST and GST fusion proteins. (B) Tfb1 and p62 bind to the TAD2 subdomain of p53. (Upper panel) GST-Tfb11–115 (1 μM) was coupled to GSH-Sepharose and incubated with 1.0 μM, 0.5 μM, and 0.1 μM of p531–73 (lanes 5–7) and 1.0 μM, 0.5 μM, and 0.1 μM of p5320–73 (lanes 8–10). In this assay, neither 1 μM of p531–73 (lane 3) nor 1 μM p5320–73 was retained by 1 μM GST coupled to GSH-Sepharose. 0.5% of 1 μM p531–73 (lane 1) and 1 μM p5320–73 (lane 2) were loaded as controls, and the p53 peptides were detected with anti-p53 antibody. (Lower panel) p5320–73 at 1.0 μM, 0.5 μM, and 0.1 μM was incubated with 1 μM GST-p621–127 (lanes 3–5) or 1 μM GST-Tfb11–115 (lanes 6–8) coupled to GSH-Sepharose. In this assay, 0.5% of p5320–73 input was loaded (lane 1) as a control, and 1 μM of p5320–73 was not retained by 1 μM GST (lane 2) bound to GSH-Sepharose. Coomassie staining verified equivalent inputs of GST and GST fusion proteins. Molecular Cell 2006 22, 731-740DOI: (10.1016/j.molcel.2006.05.007) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 The p53 TAD2 Binds to Similar Regions of the Tfb1 and p62 PH Domains (A) Ribbon model of the free p621–108 structure (Gervais et al., 2004). The residues showing chemical shift changes in the 1H-15N HSQC spectra of p621–127 upon binding to p5320–73 are shown in yellow. (B) The residues that undergo chemical shift changes in the 1H-15N HSQC spectra of Tfb11–115 upon formation of the Tfb1/p53 complex are mapped (in yellow) onto the ribbon model of the NMR structure of free Tfb1 (Di Lello et al., 2005). Molecular Cell 2006 22, 731-740DOI: (10.1016/j.molcel.2006.05.007) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Structure of the Tfb1/p53 Complex (A) Overlay of the 20 lowest-energy structures of the complex between Tfb1 (in green) and p53 (in orange). The structures were superimposed using the backbone atoms C′, Cα, and N of residues 4–65 and 85–112 of Tfb1 and residues 47–56 of p53. (B) Ribbon model for the lowest-energy conformer of the complex Tfb1 (in green)/p53 (in orange). Molecular Cell 2006 22, 731-740DOI: (10.1016/j.molcel.2006.05.007) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Structural Details of the Tfb1/p53 Interface (A) The 3D structure of Tfb1 is shown as a ribbon within the transparent molecular surface (in green), whereas the backbone trace of p53 is represented as a tube (in orange). The side chains of the three hydrophobic residues (Phe54, Trp53, and Ile50) on the amphipathic side of the p53 helix are also shown. The aromatic ring of Phe54 from p53 is positioned in a shallow pocket on the surface of Tfb1. (B) Ribbon representation of Tfb1 (in green) and backbone trace of p53 (in orange). Gln49 and Arg61 from Tfb1 form one side of the hydrophobic pocket in which the side chain of Phe54 from p53 is inserted; also, their side chains are involved in amino-aromatic and cation-π interactions with the aromatic ring of Phe54. (C) Ribbon representation of Tfb1 (in green) and backbone trace of p53 (in orange). The indole ring of Trp53 from p53 is positioned into a narrow cleft formed by the side chains of Lys57 and Met59 of Tfb1. The side chain of Trp53 (p53) contributes to the stability of the complex through a cation-π interaction with Lys57 (Tfb1) on one side of the indole ring and a sulfur-π interaction with Met59 (Tfb1) on the other side. Additional contacts between p53 and Tfb1 involve the side chain of Ile50 of p53 and both Met59 and Met88 of Tfb1. Molecular Cell 2006 22, 731-740DOI: (10.1016/j.molcel.2006.05.007) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 Effect of p53 Phosphorylation on the Complex Formation (A) Comparison of the Kd values (nM) for the binding of p62 and Tfb1 to the unphosphorylated and phosphorylated p53 peptides. (B) Selected regions of the overlay between the 2D 1H -15N HSQC spectra of 15N-labeled p621–127 in the free form (in black) and in the presence of unlabeled p5320–73 (in red). The signals undergoing significant changes in 1H and 15N chemical shifts upon formation of a 1:1 complex with p53 are highlighted by arrows. (C) Selected regions of the overlay between the 2D 1H -15N HSQC spectra of 15N-labeled p621–127 in the free form (in black) and in the presence of unlabeled p53(pS46pT55) (in red). The signals that undergo significant changes in 1H and 15N chemical shifts upon formation of a 1:1 complex with p53(pS46pT55) are highlighted by arrows. (D) Ribbon model for the lowest-energy structure of the Tfb1 (in green)/p53(pS46pT55) (in orange) complex, showing the potential ion pairs Lys11/pSer46 and Arg61/pThr55. (E) Ribbon model for the lowest-energy structure of free p621–108 (Gervais et al., 2004). The side chains of Lys18 and Lys54, which correspond to Lys11 and Gln49 in Tfb1, are also highlighted. The Tfb1/p53(pS46pT55) model and the NMR structure of free p621–108 were superimposed using the backbone atoms C′, Cα, and N of residues in secondary structure elements and were then separated for clarity. Molecular Cell 2006 22, 731-740DOI: (10.1016/j.molcel.2006.05.007) Copyright © 2006 Elsevier Inc. Terms and Conditions