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1 Supporting Online Material for Structure of PTB Bound to RNA: Specific Binding and Implications for Splicing Regulation Florian C. Oberstrass, Sigrid D. Auweter, Michèle Erat, Yann Hargous, Anke Henning, Philipp Wenter, Luc Reymond, Batoul Amir-Ahmady, Stefan Pitsch, Douglas L. Black, Frédéric H.-T. Allain* *To whom correspondence should be addressed. This PDF file includes: Materials and Methods SOM Text Figs. S1 to S4 Table S1 References Published 23 September 2005, Science 309, 2054 (2005) DOI: /science

2 Material and Methods Preparation of the protein constructs The nucleotide sequence encoding PTB1 full-length (Acc. No. X62006) was amplified by PCR and cloned into the pet-28a (+) expression vector (Novagen, Inc). The constructs PTB RBD1 (residue ), RBD2 (residue ) and RBD34 (residue ) were subcloned into the same type of vector. Expression constructs for mutant proteins PTB RBD34 H411A, H457A and the triple mutant E502K-V505E-I509K were generated using site-directed mutagenesis (QuickChange Site-Directed Mutagenesis Kit, Stratagene). All proteins contained an N-terminal His-tag and were expressed in E. coli (BL21(DE3)) by induction with 1mM IPTG. Proteins were purified by Ni-NTA affinity chromatography (Qiagen), using a step gradient of imidazole (20-500mM) for elution. Two consecutive Ni-NTA affinity chromatography steps were necessary to minimize RNase activity of the protein samples. Isotopic labeling was done by expressing the proteins in M9 medium containing 15 N-NH 4 Cl and/or 13 C-labeled glucose as the only source of carbon and nitrogen. Preparation of RNA and protein-rna complexes The unlabeled oligoribonucleotides CUCUCU, UCUCU, CUCU, CUCUCU(A) 5 CUCUCU and CUCUCU(A) 10 CUCUCU were purchased from Dharmacon Inc. (USA). CUCUCU(A) 15 CUCUCU, CUCUCU(A) 20 CUCUCU, CUCUCU(A) 25 CUCUCU and CUCUCU(A) 30 CUCUCU were synthesized in the laboratory of Prof. S. Pitsch (EPFL, Switzerland) (1). UCUCU and CUCU oligoribonucleotides containing 13 C isotopically labeled riboses were chemically synthesized as described (PW, LR, SA, FA and SP, manuscript in preparation). The protein-rna complexes were prepared either by titrating the protein into the RNA or vice versa. The titration was monitored by NMR at 303 K or 313 K. Samples in 2 H 2 O were prepared by lyophilization and resuspension in 100% 2 H 2 O. Bandshift assay were done following the same protocol as described in Amir-Ahmady et al (2). 1

3 NMR Spectroscopy NMR spectra of the complexes were acquired at 313K or 318 K on Bruker DRX-500, DRX-600 and Avance 900. All spectra were processed with XWINNMR and analyzed with Sparky 3.0 ( Triple-resonance experiments for protein backbone assignments were collected at 500 and 600 MHz on 13 C, 15 N-labeled protein in complex with unlabeled 5 CUCUCU 3 RNA in 90% H 2 O / 10% 2 H 2 O. Almost complete protein backbone and side-chain 1 H, 13 C, 15 N resonance assignment were obtained using standard experiments: HNCA, CBCA(CO)NH and HCCH-TOCSY (3). 15 N-TROSY (4), 3D 15 N- and a 13 C-edited NOESYs (τ m =150ms) were collected at 900 MHz. Resonance assignments for aromatic side chains were obtained from 2D 1 H- 1 H TOCSY and NOESY spectra collected at 900 MHz. RNA resonance assignments of the RNAs in complex with the different RBDs were achieved using 2D 1 H- 1 H TOCSY, 2D 1 H- 1 H NOESY and 2D 1 H- 1 H double-half-filtered NOESY (5) spectra collected on a 13 C, 15 N-labeled protein, in complex with unlabeled RNA, in 100% 2 H 2 O. Additionally, a 3D 13 C- edited NOESY experiment (τ m =150ms) collected on a 15 N-labeled protein in complex with RNA 13 C- labeled only on the sugars in 100% 2 H 2 O was used in order to unambiguously assign the sugar resonances. The assignments of intermolecular NOEs were based on a 3D 13 C F 1 -edited, F 3 -filtered NOESY- HSQC spectrum (τ m =150ms) (6) and a 2D 1 H- 1 H F 1-13 C-filtered F 2-13 C-edited NOESY (5) on the protein-rna complex with either the protein 13 C- 15 N labeled and the RNA unlabeled or the protein 15 N- labeled and the RNA labeled with 13 C in the sugars. Structure Calculation Seven cycles of CANDID (7) and DYANA (8) were used to generate preliminary structures and a list of automatically assigned NOE distance constraints for each protein in complex with CUCUCU (PTB 2

4 RBD1: residue ; PTB RBD2: residue ; PTB RBD34: residue ). This calculation included the peak lists from 13 C-edited NOESY, 15 N-edited NOESY and 2D homonuclear NOESY spectra. After reviewing the generated constraint lists for the protein, the complexes were calculated with DYANA (8) by adding the manually assigned intramolecular RNA and intermolecular constraint lists obtained from the 2D and 3D filtered NOESY. To generate these two latter lists, protein-rna complexes with shorter oligonucleotides needed to be prepared to avoid multiple RNA binding sites (SA, FO and FA, in preparation). For studying RBD3 and RBD4 in complex without separating the domains, we prepared two single amino acid mutations: H411A in RBD3 and H457A in RBD4. These mutations lowered the RNA binding affinity of the mutated RBD and allowed analysis of complexes with RNA bound to only one RBD (SA, FO and FA, in preparation). As with RBD1 and RBD2, the RNA needed to be shortened to prevent multiple binding registers. For all calculations of the protein- RNA complexes, random chains of RNA and protein were used as starting points. At later stages of the refinement, hydrogen-bond restraints within the protein (from slowly exchanging amides) and a few repulsive distance restraints based on the absence of NOEs (lower limit of 4Å) were used in the calculation of the complexes. For RBD1 and 2, 100 random starting structures and steps of calculation were used in DYANA (8), and for the double domain constructs RBD and respectively. Based on target function, the 40 best structures were refined by a simulated annealing run in implicit solvent in AMBER 7.0 (9) with the force field 98 for RNA. Finally, the best 20 structures based on energies, stereochemistry and Ramachandran plot were selected after analysis with MOLMOL (10) and PROCHECK (11). The statistics of these structures are listed in Table S1. All figures of molecular structures were generated with MOLMOL (10). Detailed description of the RBD-RNA interactions 3

5 PTB RBD1 interacts with three nucleotides that spread across the β-sheet surface (Fig. 1A). U 2, C 3 and U 4 are positioned on β-strands β4, β1 and β2, respectively. U 2 and C 3 interact with one another, U 2 O2 hydrogen-bonding with C 3 amino (Fig. 2A). Such intra-rna hydrogen-bonds in an RNA bound to an RBD are not frequent but were observed previously in other RBD-RNA complexes (12-14). U 2 stacks on R64, C 3 is sandwiched between H62 and N132 and U 4 is positioned in a hydrophobic pocket formed by five protein side-chains. This UCU triplet is specifically recognized, U 2 by Q129 side-chain, C 3 by S131 side-chain and by F130 and N132 main-chain and finally U 4 by the main-chain of the C-terminus (Fig. 2A). PTB RBD2 interacts very clearly with the dinucleotide C 3 U 4. C 3 and U 4 are positioned similarly as in RBD1 (Fig. 1B and 2A), with C 3 sandwiched between two side-chains and U 4 bound in a hydrophobic pocket. Both bases are sequence-specifically recognized by the main-chain of the protein and by S258. RBD2 interacts with a third nucleotide, U 6 that is not as precisely defined. U 6 is in contact with K266, K271 and Y267, all of these amino acids being located in the C-terminal extension of the RBD between β4 and β5 (Fig. 2B). The RBD2 interaction with U 6 and not to C 5 suggests that RBD2 might prefer the sequence CUU (not present in the hexamer we used here) rather than CUC. This would partly explains that CUU is often found in RNA sequences identified by SELEX (15). PTB RBD3 interacts with five nucleotides: U 2 to U 6 (Fig. 1C). Except for U 6, all the other nucleotides (U 2 to C 5 ) are well defined in the structure. RBD3 sequence-specifically recognizes U 2, C 3 and U 4 in a very similar manner as RBD1, even though only three interacting side-chains are identical (Fig.2A). Like PTB RBD2, RBD3 has an extended domain with an additional 5 th β-strand (16). However, in RBD3 this extension of the domain results in contacts with two additional nucleotides: C 5 and U 6. The base of C 5 interacts with L370 and stacks on F371 while the sugar of C 5 interacts with L426. In addition, C 5 is further stabilized by a hydrogen-bond between its amino and a phosphate oxygen of U 4. K368 4

6 contacts the phosphate oxygens of both C 5 and U 6 and P417 and R418 are in contact with U 6. (Fig. 2C). Compared to the other RBDs of PTB, RBD3 binds five pyrimidines. This explains that RBD3 was thought for a long time to have a predominant role in RNA binding (17-19). PTB RBD4 binds to three nucleotides: U 2, C 3 and U 4 (Fig. 1D). Compared to the other domains, RBD4 is interacting more tightly with U 2, since the side-chain of N448 (of the interdomain linker) stacks on the base of U 2 and is hydrogen-bonded with U 2 2 oxygen (Fig. 2A). The RBD4-RNA interactions differ from the other RBDs in how U 4 is bound. Like in the other RBDs, U 4 is in a hydrophobic pocket (contact to L495, F487 and the aliphatic parts of K485 and K489) but unlike in the other domains, U4 is flipped by 180. The unusual position of U 4 as compared to the other RBDs can be explained by a shorter C-terminal extension that prevents the formation of two hydrogen-bonds with the base of U 4 (Fig. 2A). Evidence that PTB induces RNA looping. In order to assess if PTB can indeed induce RNA looping of an RNA containing two pyrimidine-tracts, we performed binding studies of PTB34 with six different RNAs. These RNAs contained two CUCUCU hexamers, one at the 5 end and the other at the 3 end, the pyrimidine-tracts being separated by five (refered as A5 ), ten ( A10 ), fifteen ( A15 ), twenty ( A20 ), twenty-five ( A25 ) and thirty ( A30 ) adenines. When these RNAs were titrated into a solution of PTB RBD34, we observed binding in an intermediate exchange regime for all six complexes, indicating that the binding affinity is higher compared to isolated CUCUCU hexamers. At a 1 to 1 stochiometric ratio, the spectrum of the complex with A15 has sharp lines, suggesting that it is monomeric. Also, the complex with A15 has the sharpest NMR lines among all six complexes. This is consistent with our structure of the complex, as we measured that the RNA bridging the two binding sites must span a distance of 50 to 60 Å, requiring at 5

7 least 10 nucleotides. The spectrum of PTB RBD34 in a 1 to 1 stochiometric ratio with A15 is very similar to the one of RBD34 bound to two CUCUCU equivalents (Fig. S4A). This indicates that each RBD is bound to one pyrimidine tract and therefore that RNA looping is induced. With the RNA looping model, one expects that the affinity for the RNA would decrease when the distance between the pyrimidine-tracts is not optimal, either too short or too long. We found evidence in agreement with this hypothesis in the spectra of our complexes. All six complexes give almost identical spectra indicating that RBD34 bind all six RNAs effectively and similarly (Fig. S4B). However, a close inspection of several resonances that present a large chemical shift difference of their amide resonances between the free protein and the bound protein, like H414, V524 and S525 shows some small chemical shift differences (Fig. S4C and S4D). The chemical shift difference between the free and the bound protein resonances is highest in the complex with A15 and gets progressively smaller with A20, A25 and A30 (Fig. S4C and S4D). This indicates that the fraction of bound protein is the highest in the complex with A15, and decreases progressively in the complex with A20, A25 and A30, demonstrating a decrease in RNA binding affinity for PTB RBD34 when the length of the RNA linker between the pyrimidine-tracts is increased. When comparing the complex with A5 and A10, again A15 has the highest apparent affinity (Fig.S4D). In this case, five and ten nucleotides not being the optimal RNA length, the RNA binding affinity decreases possibly due to sliding of the RNA between the binding sites or formation of a dimeric complex. Furthermore, previous findings showing that RBD34 has a binding site size of 26 nucleotides (19) is consistent with our results that the 27 nucleotide RNA A15 has the highest affinity for RBD34. We also measured PTB binding to these synthetic RNAs with variable spacers by electrophoretic mobility shift assays (2). RNAs with CUCUCU elements separated by 15, 20, 25, or 30 nucleotides showed distinctly better binding to the PTB RBD34 than RNAs with 5 or 10 nucleotide spacers (Fig. 6

8 S4E). Note that the A5 RNA forms a low migrating complex at high protein concentration, suggesting formation of a dimeric complex (Fig. S4E). The affinity (in the micromolar range) was too low to measure the K d accurately, but it is clear that the protein interacts more effectively with RNAs carrying well-separated pyrimidine tracts, as predicted by the structure of our protein-rna complex. 7

9 Figure S1: 1 H- 15 N-TROSY spectra of PTB full length and of subdomains in complex with CUCUCU. A. Overlay of the 1 H- 15 N-TROSY spectra of PTB RBD1 in complex with 5 CUCUCU3 (red), PTB RBD2 in complex with 5 CUCUCU3 (green) and PTB RBD3 and 4 in complex with two equivalents of 5 CUCUCU3 (blue). B. 1 H- 15 N-TROSY spectrum of PTB full-length in complex with 4 equivalents of 5 CUCUCU 3 RNA RBD1 58 : SRVIHIRKLPI-DVTEGEVISLGLPFGKV---TNLLMLKG--KNQAFIEMNTEEAANTMVNYYTSVTPVLRGQPIYIQFSNHKELKTDS : 140 RBD2 182: -PVLRIIVENLFYPVTLDVLHQIFSKFG--TVLKIITFTKNNQFQALLQYADPVSAQHAKLSLDGQNIYNACCTLRIDFSKLTSLNVKYNND----KSRDYTRP : 278 RBD3 337: -SVLLVSNLN---PERVTPQSLFILFGVYGDVQRVKILFNK-KENALVQMADGNQAQLAMSHLNGHKLHGK--PIRITLSKHQNVQLPREGQEDQGLTKDYGNS : 433 RBD4 453: SATLHLSNIP---PSVSEEDLKVLFSSNGGVVKGFKFFQKD-RKMALIQMGSVEEAVQALIDLHNHDLGEN-HHLRVSFSKSTI : 531 IFIXNL RGFGFIXF RNP2 LYL RNP1 K YAYL Y V V V V V β1 α1 β2 β3 α1 β4 β5 Figure S2: Sequence alignment of the four RBD of PTB1. Amino acids interacting with the RNA are shown in a red box, residues in grey and black are located in the β-sheet and residues in yellow are in the α-helices. The RNP1 and RNP2 sequence consensi are shown in green. Residues in grey are pointing toward the hydrophobic core of the domain. Figure S3: 1 H- 15 N-HSQC spectra of the free PTB RBD34 (green) and RBD34 triple mutant (red) containing three side-chain changes in helix2 of RBD4 (E502K, V505E and I509K). All three side-chains contribute to the interdomain interactions. The two spectra are very different and the spectrum of the RBD34 triple mutant (green) is almost identical to the spectrum of wild-type RBD3 and RBD4 expressed individually (not shown), indicating that the two RBDs do not interact in the mutant. This independently confirms the interdomain interaction between RBD3 and RBD4 of PTB. The spectra were recorded at 900 MHz under the following conditions: 20 mm NaCl, 10mM Na-phosphate, ph=6.5, T=303K. 8

10 Figure S4: Evidence for RNA looping induced by PTB RBD34. A. Overlay of the 1 H- 15 N-HSQC spectra of PTB RBD34 in complex with one equivalent 5 CUCUCU(A) 15 CUCUCU 3 (red) and in complex with two equivalents of 5 CUCUCU3 (blue). B. Overlay of the 1 H- 15 N-HSQC spectra of PTB RBD34 in complex with one equivalent A15 (red), A20 (blue), A25 (green) and A30 (black). All four spectra were recorded at 900 MHz with a 0.25 mm sample under the following conditions: 20 mm NaCl, 10mM Na-phosphate, ph=6.5, T=313K. C. Close-up views of three selected cross-peaks of the spectra shown in B. Cross-peaks of V524 (left), S525 (center) and H411 (right) are shown. D. Plot of the proton amide chemical shift difference between the bound form and the free form of H411, V524 and S525 in complex with A5, A10, A15, A20, A25 and A30. The chemical shift difference is measured in Hz at a field strength of 900 MHz. E. Electromobility shift assay (EMSA) of PTB RBD34 with A5, A10, A15 and A20. 9

11 Supplementary Table 1 Structural Statistics of PTB RBD 1-4 a NMR Restraints RBD 1 b RBD 2 c RBD 3/4 f Distance Restraints Protein intramolecular intraresidual sequential ( i-j =1) medium range (1< i-j 4) long range ( i-j >4) hydrogen bonds a RNA intramolecular / 51 intraresidual / 44 sequential ( i-j =1) / 7 RNA-Protein intermolecular / 54 Repulsive Distance Restraints Protein RNA 2 6 Torsion Angles RNA Energy Statistics (20 Structures) NOE violations >0.3 (Å) 1.3 ± ± ± 0.3 Mean constraint violation energy (kcal mol - 1) 63.2 ± ± ± 2.9 Mean AMBER energy (kcal mol - 1) ± ± ± 27 Mean deviation from ideal covalent geometry Bond length (Å) ± 1E ± 1E ± 1E -4 Bond angle (degrees) 2.51 ± ± ± 0.02 Ramachandranplot analysis Residues in most favored regions (%) 73.2 ± ± ± 1.9 Residues in additional alowed regions (%) 21.4 ± ± ± 2.1 Residues in generously alowed regions (%) 4.2 ± ± ± 1.0 Residues in disallowed regions (%) 1.2 ± ± ± 0.8 RMSD from the mean structure (Å) RBD3 d RBD4 e Protein Backbone heavy atoms (N, C α, C', O) 0.57 ± ± ± ± 0.10 All heavy atoms 0.98 ± ± ± ± 0.10 RNA All heavy atoms 0.54 ± ± ± ± 0.16 Protein + RNA Backbone 0.58 ± ± ± ± 0.09 All heavy atoms 0.96 ± ± ± ± 0.11 Based on slow exchanging amide protons in D 2 O b RMSD is based on protein residues S58 - T138 and RNA residues U 2 -U 4 c RMSD is based on protein residues S181 - P281 and RNA residues C 3 -U 4 d RMSD is based on protein residues N336 - G431 and RNA residues U 2 -U 6 e RMSD is based on protein residues S453 - I531 and RNA residues U 2 -U 4 f RMSD of RBD3/4 protein (N336 - G431; S453 - I531) is (1.05 ± 0.18) Å, (1.49 ± 0.16) Å for the backbone and the heavy atoms, respectively 10

12 Supplementary References 1. S. Pitsch, P. A. Weiss, L. Jenny, A. Stutz, X. Wu, Helv. Chim. Acta 84, 3773 (2001). 2. B. Amir-Ahmady, P. L. Boutz, V. Markovstov, M. Phillips, L. B. Black, RNA 11, 699 (2005). 3. J. Cavanagh, W. J. Fairbrother, A. G. Palmer, N. J. Skelton, Academic Press (1996). 4. K. Pervushin, R. Riek, G. Wider, K. Wuthrich, PNAS. 94, (1997). 5. R. D. Peterson, C. A. Theimer, H. Wu, J. Feigon, J.B.NMR 28, 59 (2004). 6. C. Zwahlen et al., J.A.C.S 119, 6711 (1997). 7. T. Herrmann, P. Guntert, K. Wuthrich, J Mol Biol 319, 209 (2002). 8. P. Guntert, C. Mumenthaler, K. Wuthrich, J Mol Biol 273, 283 (1997). 9. D. A. Case et al., University of California, San Francisco. (2002). 10. R. Koradi, M. Billeter, K. Wuthrich, J Mol Graph 14, 51 (1996). 11. R. A. Laskowski, J. A. Rullmannn, M. W. MacArthur, R. Kaptein, J. M. Thornton, J. B. NMR 8, 477 (1996). 12. F. H. T. Allain, P. Bouvet, T. Dieckmann, J. Feigon, Embo J. 19, 6870 (2000). 13. C. Oubridge, N. Ito, P. R. Evans, C. H. Teo, K. Nagai, Nature 372, 432 (1994). 14. J. Ding et al., Genes Dev 13, 1102 (1999). 15. I. Perez, C. H. Lin, J. G. McAfee, J. G. Patton, RNA 3, 764 (1997). 16. M. R. Conte et al., Embo J 19, 3132 (2000). 17. Y. L. Oh et al., Biochem J 331 (Pt 1), 169 (1998). 18. A. Kaminski, S. L. Hunt, J. G. Patton, R. J. Jackson, RNA 1, 924 (1995). 19. I. Perez, J. G. McAfee, J. G. Patton, Biochemistry 36, (1997). 11