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1 Supporting Information Floor et al /pnas SI Methods Protein Expression and Purification. Schizosaccharomyces pombe Dcp1 and Dcp2 and the Saccharomyces cerevisiae Dcp1 Dcp2 complex were expressed in Escherichia coli and purified as described (1, 2). Edc1 CTR (residues ) was expressed and purified as described (3). The S. pombe Dcp1 Dcp2 complex was formed by mixing at a molar ratio for 4 h at 4 C prior to gel filtration. Isotopically labeled proteins were produced in M9 minimal media using NH 4 Cl and 13 C 2 Hor 12 C 2 H glucose as the sole nitrogen and carbon sources, respectively. Methyl labeling was achieved by addition of labeled precursors for Ile (5 mg L 1 ), Leu/Val (1 mg L 1 ), Met ( mg L 1 ), and Ala (1 mg L 1 ) 4 min prior to induction, or at an optical density of roughly.5. NMR Spectroscopy. Most NMR experiments were conducted at approximately 5 μm protein in a buffer containing mm NaCl, 1 mm Na 2 SO 4, 5 mm DTT, 21.1 mm NaH 2 PO 4, and 28.8 mm Na 2 HPO 4 (ph 7.) in H 2 OorD 2 O. Methyl side chain HCC-total correlation spectroscopy (TOCSY) experiments were acquired on a ruker 5 MHz, nitrogen heteronuclear single quantum correlations (HSQCs) on a Varian 6 MHz, methyl heteronuclear multiple quantum correlations (HMQCs) on a ruker 8 MHz, and Carr Purcell Meiboom Gill (CPMG) experiments either on ruker 8 MHz or ruker 9 MHz spectrometers. All instruments were outfitted with cryogenic probes. Sequences used were the fast-hsqc (4), 13 C-HMQC (5), 13 C- HSQC (6), relaxation compensated, constant time 13 C-CPMG (7), HCCH-TOCSY (8), (H)C(CO)NH (9), and CCH-NOESY (1). The difference in chemical shift between spectra (Fig. 5, Fig. S1, Fig. S7) was calculated using the minimum chemical shift metric, where the matched peak in the second spectrum is simply the closest (11). This metric was used because transferring assignments was unreliable because of the number of chemical shift changes. Note that this metric is conservative and will always underestimate chemical shift changes; large minimum chemical shift changes are therefore significant indicators of shifts or broadening. p Chemical shift changes were normalized according to Δδ C ¼ ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi p ðδ C 2Þ 2 þðδ H Þ 2 or Δδ N ¼ ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ðδ N 5Þ 2 þðδ H Þ 2. NMR data were processed by NMRPipe (12) and spectra were visualized using Sparky (a gift from T.D. Goddard and D.G. Kneller, University of California, San Francisco, CA). Methyl Assignment of Dcp2. De novo assignment of 73% of Dcp Ile, Leu, Val, Met, and Ala (ILVMA) methyls was completed using a divide and conquer strategy based on the nitrogen assignments of Dcp2 1 94, , and and the crystal structure of the Dcp1 Dcp2 complex [Protein Data ank (PD) ID code 2QKM]. First, methyl shifts were correlated with the backbone resonances of Dcp and using the (H) C(CO)NH. Assignments were then made based on the CCH- NOESY and predicted NOE peaks from the Dcp1 Dcp2 crystal structure chain. Predicted NOEs were calculated using an in-house modified version of MAP-XS (13) and comparison to observed NOEs was conducted manually. Assignments were confirmed if there was significant overlap between the predicted and observed NOEs and the 13 C shift agreed reasonably with the value from the (H)C(CO)NH. No interdomain NOEs were observed. Methyl assignment was completed using the Collaborative Computing Project for NMR (CCPN) suite (14) and Computer Aided Resonance Assignment (CARA) software (a gift from R. Keller, Swiss Federal Institute of Technology, Zurich, Switzerland, CPMG Data Analysis. Dispersion curves were acquired with a 4 ms constant-time delay at field strengths from 5 Hz to 95 Hz. Peaks were fit using the program FuDA (a gift from D.F. Hansen, University College London, London, UK, and intensities extracted. Intensities were converted to relaxation rates using standard procedures (7) and fit to both the fast-exchange limit (Eq. S1) () and the general Carver Richards (CR) equation for two-site exchange: R eff 2a ðν CPÞ ¼ 1 2 fr 2a þ R 2b þ k a þ k b 2ν CP cosh 1 ½D þ coshðη þ Þ D cosðη ÞŠg; where D ¼ 1 1 þ ψ þ 2Δω2 2 ðψ 2 þ ξ 2 Þ 1 2 pffiffi 2 η ¼ ½ψ þðψ 2 þ ξ 2 Þ 1 2 Š 1 2 4ν CP ψ ¼ðR 2a R 2b þ k a k b Þ 2 Δω 2 þ 4k a k b ξ ¼ 2ΔωðR 2a R 2b þ k a k b Þ [S1] and R 2a and R 2b are the transverse relaxation rates for the major and minor states in the absence of exchange, respectively, Δω is the chemical shift difference between states, k a and k b are the forward and reverse exchange rates, and ν CP is the CPMG refocusing field strength in Hz (16 18). Fitting was performed with software cpmg_fitd8 (a gift from D. Korzhnev and L. Kay, University of Toronto, Toronto, ON). The fast-exchange limit is justified based on the quality of the fits to the fast-exchange limit, statistical comparisons between fits to the fast-exchange limit and the full CR equation (Table S1, Table S2), and the shape of the dispersion curves. Three peaks (Ala6, one peak from Val11, and Ala51) were fit to the full CR equation based on two criteria: greater than 1% difference in the F-statistic of between the fast-exchange limit and CR equation fits (Table S2), and difference in fitted parameters larger than their error. Peaks for Leu74 and unassigned peak 3 both have a difference in F-statistic greater than 1%, but their fitted parameters are indistinguishable between the CR equation and fast-exchange limit fits, so they were fit to the simpler model (fast exchange). Error in R 2;eff was estimated using repeat measurements by the pooled standard deviation method (19). R 2;eff values shown for repeated points are the average of the repeats. R ex for Dcp was estimated by subtracting the 95 Hz R 2;eff from the 5 Hz R 2;eff and for Dcp from 95 Hz and Hz. CPMG Group Fitting. We used statistics, exchange rates, and mutational data to define group members. We first filtered the data using individual fits and mutational data: Individual exchange rates must be in a distribution of rate around 2;5 s 1 (Fig. S6A, excludes Ala6, one Val11 peak, Ala51, Val114 and one unassigned peak), the dynamic behavior must be severely attenuated upon mutation of Trp43 (excludes Ile12, Val112, Leu113, Val114, and Ile179), and the residue must not be dynamic in either isolated domain (excludes no new residues). These con- Floor et al. 1of1

2 straints leave fourteen residues as potential members of the group. We then fit the group to a global exchange rate and applied a statistical filter: the F-statistic for the group fit must be within % of the F-statistic for the individual fit (Table S1). This criterion did not remove any members from the group suggesting our previous filters were stringent. Kinetic Assays of Decapping. Kinetics on the S. cerevisiae Dcp1 Dcp2 complex with and without Edc1, and the S. pombe proteins, were conducted as described (2, 3, ). riefly, the S. cerevisiae and S. pombe decapping proteins were incubated with capradiolabeled 323-nucleotide MFA2 at 4 C or 29-nucleotide mrna substrates at.1 C, respectively, quenched at various time points with 5 mm EDTA and products analyzed by thinlayer chromatography. All reactions were conducted under single turnover conditions with ½EŠ ½SŠ. Structural Model of the Dcp1 Dcp2 Open Form. To generate a complete structural model of the Dcp1 Dcp2 open form, the regulatory domain from S. pombe Dcp2 alone (PD ID code 2A6T) was aligned with the regulatory domain from S. pombe Dcp2 in complex with Dcp1 (PD ID code 2QKM, chain D) using PyMOL. The resulting model was merged and used to generate the structural figure in Fig. 5. This approach was used as the catalytic domain in the open form of the Dcp1 Dcp2 structure and is missing many residues (PD ID code 2QKM, chain D). 1. Deshmukh MV, et al. (8) mrna decapping is promoted by an RNA-binding channel in Dcp2. Mol Cell 29: Floor SN, Jones N, Hernandez GA, Gross JD (1) A split active site couples cap recognition by Dcp2 to activation. Nat Struct Mol iol 17: orja MS, Piotukh K, Freund C, Gross JD (11) Dcp1 links coactivators of mrna decapping to Dcp2 by proline recognition. RNA 17: Mori S, Abeygunawardana C, Johnson MO, van Zijl PC (1995) Improved sensitivity of HSQC spectra of exchanging protons at short interscan delays using a new fast HSQC (FHSQC) detection scheme that avoids water saturation. J Magn Reson 18: Muller L (1979) Sensitivity enhanced detection of weak nuclei using heteronuclear multiple quantum coherence. J Am Chem Soc 11: Kay L, Keifer P, Saarinen T (1992) Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity. J Am Chem Soc 114: Skrynnikov NR, Mulder FA, Hon, Dahlquist FW, Kay LE (1) Probing slow timescale dynamics at methyl-containing side chains in proteins by relaxation dispersion NMR measurements: Application to methionine residues in a cavity mutant of T4 lysozyme. J Am Chem Soc 123: Kay L, Xu G, Singer A, Muhandiram D, Forman-Kay J (1993) A gradient-enhanced HCCH-TOCSY experiment for recording side-chain H-1 and C-13 correlations in H2O samples of proteins. J Magn Reson 11: Gardner KH, Konrat R, Rosen MK, Kay LE (1996) An (H)C(CO)NH-TOCSY pulse scheme for sequential assignment of protonated methyl groups in otherwise deuterated () N, (13)C-labeled proteins. J iomol NMR 8: Muhandiram D, Farrow N, Xu G-Y, Smallcombe SH, Kay LE (1993) A gradient 13C NOESY-HSQC experiment for recording NOESY spectra of 13C-labeled proteins dissolved in H2O. J Magn Reson 12: Farmer T, et al. (1996) Localizing the NADP+ binding site on the Mur enzyme by NMR. Nat Struct iol 3: Delaglio F, et al. (1995) NMRPipe: A multidimensional spectral processing system based on UNIX pipes. J iomol NMR 6: Xu Y, et al. (9) Automated assignment in selectively methyl-labeled proteins. JAm Chem Soc 131: Vranken WF, et al. (5) The CCPN data model for NMR spectroscopy: Development of a software pipeline. Proteins 59: Allerhand A, Thiele E (1966) Analysis of Carr Purcell spin-echo NMR experiments on multiple-spin systems. II. The effect of chemical exchange. J Chem Phys 45: Carver J, Richards R (1972) General 2-site solution for chemical exchange produced dependence of T2 upon Carr Purcell pulse separation. J Magn Reson 6: Davis DG, Perlman ME, London RE (1994) Direct measurements of the dissociationrate constant for inhibitor-enzyme complexes via the T1 rho and T2 (CPMG) methods. J Magn Reson 14: Tollinger M, Skrynnikov NR, Mulder FA, Forman-Kay JD, Kay LE (1) Slow dynamics in folded and unfolded states of an SH3 domain. J Am Chem Soc 123: Demers J-P, Mittermaier A (9) inding mechanism of an SH3 domain studied by NMR and ITC. J Am Chem Soc 131: Jones N, Quang-Dang D-U, Oku Y, Gross JD (8) A kinetic assay to monitor RNA decapping under single-turnover conditions. Methods Enzymol 448:23 4. Floor et al. 2of1

3 A 1-94 (regulatory domain) A vs WT 1-94 vs W43A Chemical Shift Change (ppm) L59 Residue Unass (catalytic domain) G vs WT vs W43A Chemical Shift Change (ppm) G168 I175, I176 F2 I13 Residue Unassigned Fig. S1. Mutation of Trp43 decouples the two domains of Dcp Shown is the chemical shift change between the isolated regulatory domain (A; residues 1 94) or catalytic domain (; residues ) and Dcp (red) or Dcp Trp43Ala (black). Chemical shift changes between the isolated domains and two-domain constructs that are outside the linker region ( 9 1) are indicative of interdomain interactions. Floor et al. 3of1

4 13 C (ppm) A C D 1 13 C (ppm) 13 C (ppm) C (ppm) A17 A66 A6 A41 A51 A239 A A6 A41 A51 A239 A A17 A66 A6 A41 A51 A239 A I I I I193 I179 I175 I179 I175 I179 I175 I238 I238 I238 I223 I223 I223 I6 I12 I6 I12 I6 I12 I13 I13 I13 I233 I49/I141 I233 I49/I141 I233 I49/I141 Ref. I196 Ref. Ref. Ref. I196 5 Hz A17 A66 5 Hz 5 Hz 5 Hz V137 V137 V137 L56 V63 L56 V63 L56 V63 L229 V137 L229 V137 L229 V137 V144 V144 V144 V11 V11 V11 V63 V63 V63 A11 A11 A11 L1 V11 L74 V11 L74 V11 L74 L L14 L34 L L14 L34 L L14 L34 L74 L74 L74 V144 L181 V144 L181 V144 L21 L21 L21 L181 L68 L34 L68 L34 L68 L34 L9 L9 L9 L1 L1 L1 L229 L229 L229 L4 L4 L4 L229/ L229/ L229/ L59 L59 L59 L14 L14 L14 L L L L56 L56 L56 I Hz 95 Hz 95 Hz 95 Hz H (ppm) 1 H (ppm) 1 H (ppm) Fig. S2. Example of CPMG spectra taken from 43 μmdcp at 9 MHz. The reference spectrum with no CPMG element (black), 5 Hz refocusing field strength (blue), and 95 Hz are shown. Many, but not all, assignments are indicated for clarity. (A) The entire methyl region. () The alanine region. (C) The isoleucine region. (D) The leucine and valine region. Floor et al. 4of1

5 3 1 Val11 Leu34 4 Ala41 3 Val , , , , 3 Leu Leu9 3 Ile12 3 Val , , , 3 Leu113 Val114 Ile13 Val , R 2,eff (s -1 ) , 4 Ile , 3 Ile , 1 Unass.(1) , Unass.(2) , , , , 5 4 Unass.(3) 5 4 Unass.(4) 6 5 Unass.(5) 5 4 Unass.(6) , , , , Ala6 Val11 Ala , , ν CPMG (Hz) , Fig. S3. A comparison of the CPMG dispersion curves at 43 μm and μm concentration of Dcp at 9 MHz. High concentration data is in red and low concentration is in green. The increased scatter of the data at low concentration is likely due to the decreased signal to noise, as both experiments were acquired with the same number of transients. Assignments are indicated in the upper right or Unass for unassigned. Floor et al. 5of1

6 A 3 1 Val Leu Ala Val , , , , 4 3 Leu Leu9 4 3 Ile Val , , , , 5 4 Leu Val Ile Val R 2,eff (s -1 ) , 5 Ile , 4 Ile , Unass. (1) , 4 3 Unass. (2) , , , , R 2,eff (s -1 ) C R 2,eff (s -1 ) , , 5 Unass. (3) Unass. (4) Unass. (5) Unass. (6) Ala , , ν CPMG (Hz) 3 Val , ν CPMG (Hz) 4 3 Ala , Leu68 Ile162 Ile , , , ν CPMG (Hz) , Fig. S4. All fitted CPMG data are shown. In each plot red is at 9 MHz and black is at 8 MHz. Assignments are indicated in the upper right or Unass for unassigned. (A) Solid lines are fits to the fast-exchange limit (Eq. 1). Errors in all panels are pooled standard deviation (SI Methods). Fit parameters are in Table S1. () Solid lines are fits to the full CR equation (SI Methods). (C) Shown are three additional dispersion curves that were not quantified because of excessive scatter at 9 MHz. Leu68, Ile162, and Ile233 peaks are shown with 9 MHz data in red and 8 MHz data in black. Floor et al. 6of1

7 A 1-94 (regulatory domain) R ex (s -1 ) Unassigned (catalytic domain) R ex (s -1 ) Residue Unassigned Fig. S5. The contribution to R 2 from chemical exchange (R ex ) for the regulatory and catalytic domains of Dcp2 is shown. R ex is calculated as the difference between low and high CPMG repetition rates. Peaks with R ex > 5 are considered dynamic. Residue assignments are indicated. (A) Dynamics in the isolated regulatory domain. () Dynamics in the isolated catalytic domain. 11 N (ppm) H (ppm) Fig. S6. Mutation of Arg167 to glutamine does not affect the ligand-free state of Dcp2. Superimposed are the nitrogen HSQCs of wild-type spdcp2 (red) and Arg167Gln (cyan). Small shifts are likely due to mutation but comparison to Fig. 1 makes it clear that Arg167Gln is not altering conformational dynamics of Dcp2. Arg167interacts withtrp43 in the closed crystal structure ofdcp2. The signal tonoise ofthe Arg167Gln HSQCis lowbecause the protein concentration is only 5 μm. Floor et al. 7of1

8 A Count ,- 1,5-2,- 2,5-3,- 3,5-4,- 4,5-1, 1,5 2, 2,5 3, 3,5 4, 4,5 5, k ex in 3 1 Val Leu Ala Val , , , , 4 3 Leu Leu9 4 3 Ile Val R 2,eff (s -1 ) , 4 Ile , , , 6 5 Unass. (2) Unass. (3) Unass. (4) , , , , Unass. (5) Unass. (6) , , ν CPMG (Hz) Fig. S7. (A) A binned histogram of fitted CPMG exchange rates is shown. For each rate bin of size 5 s 1 the count of fitted rates from Table S1 that fall in this bin is plotted. ins used for group fitting are 1,5 2,, 2, 2,5, 2,5 3,, and 3; 3;5 s 1.() Fitted CPMG data for members of the fourteenresidue group are shown. In each plot red is at 9 MHz, black is at 8 MHz, solid lines are fits to the fast-exchange limit (Eq. 1), and points are experimental data. Errors are pooled standard deviation (see SI Methods). Assignments are indicated in the upper right or Unass for unassigned. Each fit only has one free parameter in this case as the exchange rate is fit globally. Floor et al. 8of1

9 A 1 spdcp2 D1D2 WT D2 W43A D1D2 W43A I233 I C (ppm) V114 V112 L L74 L21 L L1 D1D2 WT D2 W43A D1D2 W43A Chemical Shift Change (ppm) H (ppm) Residue Fig. S8. Dcp1 causes shifts on Dcp2 remote from its binding site, some of which are blocked by Trp43Ala mutation. (A) Four ILVMA methyl HMQC spectra are superimposed: Dcp (red), Dcp1 Dcp (orange), Dcp2 Trp43Ala (black), and Dcp1 Dcp2 Trp43Ala (gray). A subset of residues is indicated that shift upon addition of Dcp1. () The minimum chemical shift change between wild-type Dcp and three other different samples are shown: Dcp1 Dcp2 (orange), Dcp2 Trp43Ala (black), and Dcp1 Dcp2 Trp43Ala (gray). Unassigned residues are not displayed for clarity. Floor et al. 9of1

10 Table S1. The CPMG fit parameters for the fast-exchange limit equation (Eq. 1) Residue k ex (s 1 ) Φðp A p Δω 2 Þ (ppm 2 ) Grp k ex (s 1 ) Group Φ (ppm 2 ) F grp F indiv F fast F CR Val11 1,7 ± 238 ð þ 1 3 2,299 ± 74 ð þ Leu34 2,285 ± 31 ð þ 1 2 2,299 ± 74 ð þ Ala41 2,492 ± 487 ð2.11.5þ 1 2 2,299 ± 74 ð þ Val63 1,766 ± 227 ð Þ 1 3 2,299 ± 74 ð þ Leu74 3,324 ± 676 ð þ 1 2 2,299 ± 74 ð1.4.1þ Leu9 2,22 ± 34 ð þ 1 2 2,299 ± 74 ð þ Ile12 2,1 ± 234 ð1.98.þ Val112 2,34 ± 29 ð þ Leu113 2,858 ± 411 ð þ Val114 4,271 ± 1896 ð þ Ile13 2,795 ± 286 ð þ 1 2 2,299 ± 74 ð þ Val144 2,162 ± 474 ð Þ 1 3 2,299 ± 74 ð þ Ile179 2,598 ± 164 ð þ Ile196 2,3 ± 18 ð þ 1 2 2,299 ± 74 ð2.1.83þ Unass (1) 1,329 ± 192 ð5..65þ Unass (2) 2,228 ± 324 ð1.2.17þ 1 2 2,299 ± 74 ð1.6.þ Unass (3) 2,587 ± 129 ð þ 1 2 2,299 ± 74 ð þ Unass (4) 3,2 ± 527 ð þ 1 2 2,299 ± 74 ð2.8.24þ Unass (5) 2,651 ± 2 ð5..54þ 1 2 2,299 ± 74 ð þ Unass (6) 2,383 ± 11 ð þ 1 2 2,299 ± 74 ð þ Fits to the fast-exchange limit of the Carver Richards equation (Eq. 1). Unassigned (Unass) residues appear in the order they are displayed in Fig. S4. For each residue the exchange rate (k ex ), Φ-parameter (p A p Δω 2 ), group exchange rate, group Φ, the ratio of the F-statistic for the group and individual fits (F grp F indiv ), and the ratio of the F-statistic for the fast-exchange limit and full Carver Richards equation fits (F fast F CR ) are given. Errors are standard error of the fit parameter. Table S2. The CPMG fit parameters for the full Carver Richards equation (Eq. S1) Residue k ex (s 1 ) p Δω (ppm) F fast F CR Ala6 79 ± 278 ð þ ± Val ± 364 ð Þ ± Ala ± 286 ð þ ± Fits to the full Carver Richards equation (Eq. S1). For each residue the exchange rate (k ex ), minor-state population (p ), chemical shift change (Δω), and the ratio of the F-statistic for the fast-exchange limit and full Carver Richards equation fits (F fast F CR ) are given. The full Carver Richards equation was used rather than the fast-exchange limit when two criteria were met: the F-statistic ratio F fast F CR showed more than a 1% difference, and the fitted parameters between the fast-exchange limit and full Carver Richards fits were separated by more than their error. The only peaks that meet these two criteria are assigned to Ala6, Val11, and Ala51 Floor et al. 1 of 1