Supplemental Data. Structure of the Rb C-Terminal Domain. Bound to E2F1-DP1: A Mechanism. for Phosphorylation-Induced E2F Release

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1 Supplemental Data Structure of the Rb C-Terminal Domain Bound to E2F1-DP1: A Mechanism for Phosphorylation-Induced E2F Release Seth M. Rubin, Anne-Laure Gall, Ning Zheng, and Nikola P. Pavletich Section 1

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3 Representative isothermal calorimetry data used to determine binding constants for the protein and peptide interactions discussed in the text. The interaction corresponding to each data set is noted on each figure. Data was acquired and analyzed as described in Experimental Procedures.

4 Section 2 Heteronuclear single quantum correlation (HSQC) spectra for (A) 15 N labeled RbC , (B) 15 N labeled E2F1 CM alone and (C) 15 N labeled E2F1 CM -unlabeled DP1 CM. The lack of 15 N and particularly 1 H chemical shift dispersion in (A) and (B) are consistent with polypeptides lacking significant structure (Dyson and Wright, 2004). The fact that the NMR data suggests that E2F1 CM is unstructured in the absence of DP1 CM is consistent with the structural and biochemical studies presented in this study, which indicate that the stability of E2F1 CM requires heterodimerization. Proteins were expressed and purified as described in Experimental Procedures except E2F and DP were expressed separately and E. coli were grown using M9 minimal media with 1 g/l 15 N ammonium chloride as the sole nitrogen source. HSQC spectra were acquired with a 600 MHz Varian spectrometer at 30 C using a gradient-enhanced pulse sequence (Kay et al., 1992). Samples consisted of mm protein in a buffer containing 50 mm sodium phosphate, 100 mm NaCl, 2 mm DTT, ph 6.1.

5 Section 3 Most of the intermolecular interactions in the coiled-coil involve non-interchangeable residues of E2F1 and DP1. In the canonical portion of the coiled coil, E2F residues 203 to 224 have a sequence reminiscent of the leucine zipper hydrophobic repeat, with leucine residues at the d position (Leu206, Leu213 and Leu220) and generally hydrophobic residues at the a position (Leu203, Leu210 and Glu217) (Lupas, 1996). By contrast, the sequence of DP1 is significantly different from that of a leucine zipper. The d position is occupied by Leu205, Arg212 and Lys219, and the a position has Cys202, Arg209 and Leu216. In addition, the acidic and basic residues that form stabilizing salt bridges are segregated to E2F1 and DP1 respectively (salt bridges between Asp209 (g), Glu217 (a), and Asp221 (e) of E2F1 and Arg209 (a), Arg215 (g), and Lys219 (d) of DP1). Section 4 Sequence alignment of p107 and p130 orthologs. The sequences used in the alignment are p107 human (hs), p107 mouse (mm), p107 chicken (gg), p130 human (hs), p130 mouse (mm), p130 rat (rn), p130 chicken (gg), and p130 fugu (fr). Conservation is indicated in cyan, and putative phosphorylation sites are also marked. The predicted secondary structure was determined using the PredictProtein Server and software therein (Rost et al., 2003). Section 5 The study of Dick et al. assayed for RbC binding in the presence of extracts from cells transfected with HA-E2F-DP (Dick and Dyson, 2003). It is thus conceivable that their E2F-DP or GST-RbC proteins might have been differentially phosphorylated or associated with endogenous factors that affected their ability to interact. It is also possible that the boundaries of the various protein fragments used contribute to the differences in the two studies. We note that the RbC Dick et al. used starts at residue 792 and is thus missing half of the RbC nter motif that is required for high affinity E2F-DP binding, although our data shows that the RbC core, which is intact in their experiments, has comparable affinity for both E2F1 and E2F4. Conversely, the E2F1(1-374) fragment used in the Dick et al. study contains ~74 additional residues C-terminal to the E2F1 CC-MB structure, which ends at residue 300, raising the possibility that the region contains a third Rb-binding element that is indeed unique to E2F1. In this respect, we note that this region has two evolutionarily conserved sequence blocks embedded in an otherwise low-complexity and lowconservation sequence. Section 6 E2F1 CM -DP1 CM -RbC crystals form in space group C222 1 with a = 146.8, b = 168.6, and c = 48.3 Å and contain one ternary complex in the asymmetric unit. The selenomethionine-substituted complex was expressed using an E. coli methionine auxotroph (B834[DE3] Novagen) in minimal media supplemented with 50 mg/l selenomethionine. Crystals were flash frozen in liquid nitrogen for data collection in crystallization buffer containing 25% v/v ethylene glycol. Multiwavelength anomalous diffraction (MAD) data sets were collected at the X4A beamline of the National Synchrotron Light Source (Brookhaven National Laboratories), and a final high-resolution native data set was collected at the ID-24 beamline of the Advance Photon Source (Argonne National Laboratories). Data were processed with DENZO and SCALEPACK (Otwinowski and Minor, 1997) and MAD phases were calculated at 3.0 Å using SHARP (Bricogne et al., 2003). The model was built with O (Jones et al., 1991) and refined with CNS (Brunger et al., 1998) and REFMAC using TLS refinement (CCP4, 1994).

6 Supplemental References CCP4 (Collaborative Computational Project 4). (1994). The CCP4 suite: programs for protein crystallography. Acta Cryst D 50, Bricogne, G., Vonrhein, C., Flensburg, C., Schiltz, M., and Paciorek, W. (2003). Generation, representation and flow of phase information in structure determination: recent developments in and around SHARP 2.0. Acta Cryst D 59. Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J.-S., Kuszewski, J., Nilges, N., Pannu, N. S., et al. (1998). Crystallography and NMR system (CNS): A new software system for macromolecular structure determination. Acta Cryst, D 54. Dick, F. A., and Dyson, N. (2003). prb contains an E2F1-specific binding domain that allows E2F1-induced apoptosis to be regulated separately from other E2F activities. Mol Cell 12, Dyson, H. J., and Wright, P. E. (2004). Unfolded proteins and protein folding studied by NMR. Chem Rev 104, Jones, T. A., Zou, S. W., Cowan, S. W., and Kjeldgaard, M. (1991). Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystal D 54, Kay, L., Keifer, E. P., and Saarinen, T. (1992). Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity. J Am Chem Soc 114. Lupas, A. (1996). Coiled coils: new structures and new functions. Trends Biochem Sci 21, Otwinowski, Z., and Minor, W. (1997). Processing of X-ray Diffraction Data Collected in Oscillation Model. In Methods in Enzymology, J. Carter, C.W., and R. M. Sweet, eds. (Academic Press), pp Rost, B., Yachdav, G., and Liu, J. (2003). The PredictProtein Server. Nuc Acids Res 32, W321-W326.