Supplementary Figure 1 Definition and assessment of ciap1 constructs. (a) ciap1 constructs used in this study are shown as primary structure schematics with domains colored as in the main text. Mutations and deletions are indicated with magenta lines. The mutations in the ciap1-b3r construct reduce proteolysis and non-native disulfide
formation 1. (b) The binding of ciap1-b3r (blue) and ciap1-b3r MF-AA (gray) to monobiotinylated ubiquitin was tested using biolayer interferometry. Equilibrium response values are plotted against ciap1 concentration. No detectable binding is observed by the ciap1- B3R MF-AA construct. ciap1-b3r binds ubiquitin with a K D of approximately 20 M as determined by a single-site binding isotherm fit, in good agreement with past studies 2. Recent reports have suggested that this mutation destabilizes ciap1 3, which might contribute to the very slight decrease in E2 binding affinity we observe in the MF-AA variants. The use of ciap1 MF-AA in E2 binding studies removes the complicating effects of direct ciap1-ubiquitin binding in the case of the E2-Ub conjugate. (c) The binding of ciap1-b3r (blue) and ciap1-b3r- CARD (green) to E2 SRCK was assessed using biolayer interferometry. Equilibrium response values were plotted as a function of E2 concentration, and the resulting curves were fit to a single-site binding isotherm. The affinities determined are approximately 27 M (ciap1-b3r) and 19 M (ciap1-b3r- CARD), recapitulating the relative increase in affinity upon CARD deletion observed in the case of ciap1-b3r MF-AA.
Supplementary Figure 2 UbcH5c does not induce dimerization of ciap1.
(a) ciap1-b3r responds to SMAC mimetics by dimerizing, as detected by native gel electrophoresis. Lane 1 shows a native molecular weight marker (Native Mark, Invitrogen) with molecular weights indicated at left. Lane 2 shows monomeric ciap1 (64 M). Lane 3 shows dimerized ciap1 in the presence of AVPW (1 mm). Binding of the bivalent SMAC mimetic BV6 (1 mm) induces stronger and more compact dimerization because it can engage two BIR3 domains simultaneously. Lanes 4-7 demonstrate that UbcH5c (64 M) does not affect the dimerization state of ciap1. Lanes 8-10 demonstrate that the gel mobility of UbcH5c is not affected by the SMAC mimetics. All samples contain 1% DMSO. (b) ciap1-b3r (64 M) was incubated with UbcH5c at 0 to 128 M. No detectable dimer is formed. ciap1 bound to BV6 is included as a dimerization control.
.
Supplementary Figure 3 Methionine methyl groups assigned by mutagenesis. 1 H- 13 C HMQC spectra of each methionine to leucine point mutant (blue) overlaid with the ciap1-b3r spectrum (red). The M466L mutant is also shown with the contours drawn four times lower. The M266L, M391L, M392L, and M402L spectra display secondary chemical shift changes that may be due to structural perturbations as a result of the mutation.
Supplementary Figure 4 Dispersion profiles of UBA α3. The residues in UBA α3 were fit to a single exchange process with k ex = 970 ± 100 s -1 and p B = 2.2 ± 0.1 %. Data and fits at 900 MHz are shown in red, 800 MHz data and fits are in blue. Error bars were determined by treating the spectral noise level as the uncertainty in peak heights. The two residues in UBA α3 that do not have detectable R 2 dispersion, Q411 and L418, are severely overlapped in the 1 H- 15 N spectra. As shown in Fig. 4a, R 2 dispersion was also detected at additional residues in the UBA (A388, V389, M390, A399, F404, K425, D429 and I430) as well as at five additional residues outside the UBA domain (L359, T534, I548, V581 and I608);
however, as the residues do not cluster into large, contiguous surfaces we did not fit the data to extract kinetic or equilibrium parameters describing the exchange processes.
Supplementary Figure 5 Characterization of the conformational states of ciap1 variants. (a) Size-exclusion multi-angle light scattering (SEC-MALS) traces of ciap1 constructs with and without SMAC mimetics. Apo-proteins are shown in black, AVPW-bound protein in blue, and BV6 bound proteins (as a dimerization control) in red. The UV absorbance for each peak was normalized to 1. As expected, ciap1-b3r (top) dimerizes in response to both AVPW and BV6, while ciap1-b3r- CARD (center) is constitutively open and dimerizes only in the presence of BV6. The L617E construct (bottom) blocks AVPW-induced dimerization without disrupting the closed conformation. (b) L617 is positioned in the RING dimerization interface. The structure of the
ciap2 RING dimer (PDB code: 3EB5, ref. 4) is shown in cartoon representation with the homologous residue to L617, L603, colored magenta and shown as spheres. Zinc ions are shown as light gray spheres.
Supplementary Figure 6 Standard SAXS models. Averaged, filtered SAXS models from 10 independent ab initio calculations are shown for ciap1-b3r, ciap1-b3r- C7 and ciap1- B3R L617E with and without AVPW. Fits (dark blue lines) of the raw scattering data (red circles) to the best ab initio model from each set are shown. Chi values for those fits are also shown. Models were generated as described (see Methods) using GASBOR.
Supplementary Figure 7 Molecular models based on standard ciap1 SAXS data. (a) The best of ten molecular models of ciap1-b3r- C7 is shown superposed with the averaged, filtered ab initio model. A normalized spatial discrepancy (NSD) for the aligment is indicated. The fit (dark blue line) of the molecular model to the experimental data (red circles) is shown below the model, and the Chi value describing the fit is indicated. I stands for scattering intensity and q is proportional to the scattering angle (q = 4 sin( )/, where 2 = the angle between the incident X-ray beam and the detector, and = the X-ray wavelength in Ångstroms). (b) The same data as in (a) is shown for ciap1-b3r- C7 in the presence of 1 mm AVPW. (c, d) Constraints used in the generation of closed monomeric molecular models are shown. All constraints are based on mutational data 1. (e) The best of ten molecular models of ciap1-b3r is shown superposed with the averaged, filtered ab initio model, in two views. The NSD between the two models is indicated. (f) The fit of the best ciap1-b3r molecular model to the experimental data is shown. All colors and variables are as in (a). (g) A subset of calculated molecular models for ciap1-b3r is shown aligned by their BIR3 domains to demonstrate the heterogeneity of the position of the CARD. The protein likely exists as an ensemble of states not fully reflected by any single model. (h) The distributions of R g values in the ensembles of molecular models generated by EOM for ciap1-b3r (black), ciap1- B3R L617E (orange), ciap1-b3r- C7 (violet) and ciap1-b3r L617E + AVPW (green) are shown. The initial, unoptimized pools are displayed as dotted lines, and the optimized ensembles as solid lines. Note that the closed states (ciap1-b3r and ciap1-b3r L617E ) adopt tighter and smaller distributions than the open states (ciap1-b3r- C7 and ciap1-b3r L617E + AVPW), reflecting the more rigid conformation of the closed states.
Supplementary Figure 8
Controls for TR-SAXS measurements. (a,b) Time-resolved SAXS (TR-SAXS) experiments of ciap1-b3r and ciap1-b3r L617E mixed with buffer instead of AVPW. (c, d) TR- SAXS experiment of ciap1-b3r- C7 mixed (c) with buffer and (d) with AVPW. (e) TR-SAXS analysis of ciap1-b3r L617E with a higher concentration of AVPW (1 mm). The relative populations of the monomeric, open, and dimeric conformations of ciap1 were extracted from the TR-SAXS data by deconvolution using the static scattering curves of each state as reference (See Methods). Each point represents and average from three experiments, plus or minus standard deviation. Data are fit to first order integrated rate equations. The closed monomer fraction is shown as blue squares, open fraction as hot pink circles, dimer fraction as purple diamonds.
Supplementary Tables Supplementary Table 1. Kinetic constants for the association of ciap1 with E2 and E2- Ub. E2 type: E2SRCK E2SRCK- Ub Construct AVPW koff (s - 1 ) kon (M - 1 s - 1 ) koff (s - 1 ) kon (M - 1 s - 1 ) ciap1- B3RMF- AA 0.78 ± 0.09 12,000 ± 2,000 0.069 ± 0.009 150,000 ± 20,000 ciap1- B3RMF- AA- ΔCARD 0.8 ± 0.2 25,000 ± 5,000 0.048 ± 0.008 220,000 ± 40,000 ciap1- B3RMF- AA + 0.51 ± 0.04 15,000 ± 7,000 0.043 ± 0.002 320,000 ± 50,000 ciap1- B3RMF- AA- ΔCARD + 0.6 ± 0.1 20,000 ± 7,000 0.040 ± 0.003 400,000 ± 100,000 Off- rates were measured directly from the dissociation phases of biolayer interferometry experiments. Reported values are the averages and standard deviations of three independent experiments. The association phases were too rapid to make meaningful direct measurement of kon values, and so the on- rates reported here are calculated from the measured koff values and the measured equilibrium KD values. Supplementary Table 2. Standard SAXS statistics. ciap1- B3R ciap1- B3RL617E ciap1- B3R- ΔC7 AVPW + + + MWSAXS (MWactual) (kda) 40.8 (39.2) 80.2 (78.5) 40.8 (39.2) 47.9 (39.2) 41.5 (38.4) 40.5 (38.4) Rg (Å) 27.4 44.6 29.0 36.8 36.4 36.6 Dmax (Å) 85.0 175.0 100.0 130.0 130.0 130.0 Porod volume 64484 105950 58413 65467 55260 52762 (Å 3 ) χ 2 (GASBOR, mean) 1.60 ± 0.09 1.5 ± 0.2 1.19 ± 0.08 1.39 ± 0.06 1.4 ± 0.2 1.4 ± 0.1 χ 2 (GASBOR, best) 1.47 1.22 1.11 1.33 1.13 1.20 NSD(DAMAVER, mean) 1.10 ± 0.03 1.6 ± 0.1 1.07 ± 0.04 1.40 ± 0.08 1.33 ± 0.04 1.30 ± 0.07 χ 2 (BUNCH, mean) 3.0 ± 0.3 ND ND ND 1.21 ± 0.04 1.3 ± 0.1 χ 2 (BUNCH, best) 2.65 ND ND ND 1.13 1.11 All reported statistics are from 1 mg/ml samples. Values are calculated using the listed ATSAS programs. SAXS- derived molecular weights were calculated using SAXS MoW 3. ND = not determined. Where errors are reported, values reflect averages of ten independent calculations and errors reflect standard deviations.
Supplementary Table 3: EOM statistics Construct AVPW Avg. Rg (Å) initial pool Avg. Rg (Å) optimized ensemble Avg. NSD optimized ensemble (DAMVER) χ 2 (EOM) ciap1- B3R 36.4 29.4 1.7 ± 0.4 1.21 + ND ND ND ND ciap1-36.2 30.8 1.9 ± 0.4 1.01 B3RL617E + 36.9 36.6 2.2 ± 0.3 1.36 ciap1- B3R- 36.3 37.1 2.1 ± 0.3 1.05 ΔC7 + 36.3 38.1 2.4 ± 0.3 0.949 Chi values and average Rg values (for the random initial pools and optimized ensembles) were returned by EOM. Also shown are average NSDs of the final optimized pools (n = 20 ± s.d.), calculated using DAMAVER. Larger NSDs are consistent with more flexible systems. ND=not determined.
Supplementary References 1. Dueber, E. C. et al. Antagonists induce a conformational change in ciap1 that promotes autoubiquitination. Science 334, 376 380 (2011). 2. Dynek, J. N. et al. c- IAP1 and UbcH5 promote K11- linked polyubiquitination of RIP1 in TNF signalling. EMBO J. 29, 4198 4209 (2010). 3. Budhidarmo, R. & Day, C. L. The Ubiquitin- Associated Domain of Cellular Inhibitor of Apoptosis Proteins Facilitates Ubiquitylation. J. Biol. Chem. in press (2014). 4. Mace, P. D. et al. Structures of the ciap2 RING domain reveal conformational changes associated with ubiquitin- conjugating enzyme (E2) recruitment. J. Biol. Chem. 283, 31633 31640 (2008).