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Internal motions prime cIAP1 for rapid activation

Nature Structural & Molecular Biology volume 21, pages 1068–1074 (2014)Cite this article

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Abstract

Cellular inhibitor of apoptosis 1 (cIAP1) is a ubiquitin ligase with critical roles in the control of programmed cell death and NF-κB signaling. Under normal conditions, the protein exists as an autoinhibited monomer, but proapoptotic signals lead to its dimerization, activation and proteasomal degradation. This view of cIAP1 as a binary switch has been informed by static structural studies that cannot access the protein's dynamics. Here, we use NMR spectroscopy to study micro- and millisecond motions of specific domain interfaces in human cIAP1 and use time-resolved small-angle X-ray scattering to observe the global conformational changes necessary for activation. Although motions within each interface of the 'closed' monomer are insufficient to activate cIAP1, they enable associations with catalytic partners and activation factors. We propose that these internal motions facilitate rapid peptide-induced opening and dimerization of cIAP1, which undergoes a dramatic spring-loaded structural transition.

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Figure 1: E2 binding to monomeric and dimeric cIAP1.
Figure 2: AVPW binding kinetics.
Figure 3: NMR spectra.
Figure 4: R2 dispersion data.
Figure 5: Standard SAXS data of closed, open and dimeric forms of cIAP1.
Figure 6: Monitoring peptide-induced conformational changes of cIAP1 by TR-SAXS.

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Acknowledgements

We thank R. Klevit for helpful discussions and A. Taherbhoy for assistance in preparing ubiquitin-charged E2. We acknowledge use of the Central California 900-MHz Facility (supported by US National Institutes of Health (NIH) grant GM68933) and thank D. Wemmer and J. Pelton for assistance with the facility. Use of the Stanford Synchrotron Radiation Lightsource (SSRL), Stanford Linear Accelerator Center (SLAC) US National Accelerator Laboratory, is supported by the US Department of Energy (DOE), Office of Science, Office of Basic Energy Sciences under contract no. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the NIH National Institute of General Medical Sciences (NIGMS) (including grant P41GM103393). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH.

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Author notes

  1. Aaron H Phillips & John W Blankenship

    Present address: Present addresses: Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA (A.H.P.) and Department of Molecular Biology and Protein Engineering, Emergent BioSolutions, Seattle, Washington, USA (J.W.B.).,

  2. Aaron H Phillips and Allyn J Schoeffler: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Early Discovery Biochemistry, Genentech, South San Francisco, California, USA

    Aaron H Phillips, Allyn J Schoeffler, John W Blankenship, Kerry Zobel, Erin C Dueber & Wayne J Fairbrother

  2. Stanford Synchrotron Radiation Lightsource, Stanford Linear Accelerator Center National Laboratory, Menlo Park, California, USA

    Tsutomu Matsui & Thomas M Weiss

  3. Department of Biochemical and Cellular Pharmacology, Genentech, South San Francisco, California, USA

    Anthony M Giannetti

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Contributions

A.H.P. designed and performed NMR experiments, analyzed data and wrote the paper. A.J.S. designed and performed SAXS and biochemical experiments (except surface plasmon resonance experiments), analyzed data and wrote the paper. T.M. and T.M.W. designed the specialized stopped-flow SAXS instrumentation and performed SAXS experiments. K.Z. prepared peptide reagents. A.M.G. performed surface plasmon resonance experiments. J.W.B. performed selected NMR experiments. E.C.D. and W.J.F. supervised the work, designed experiments and contributed to the writing of the paper.

Corresponding authors

Correspondence to Erin C Dueber or Wayne J Fairbrother.

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Competing interests

A.J.S., A.M.G., K.Z., E.C.D. and W.J.F. are all employed by Genentech, a member of the Roche group. J.W.B. is employed by Emergent Biosolutions.

Integrated supplementary information

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 formation1. (b) The binding of cIAP1-B3R (blue) and cIAP1-B3RMF-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-B3RMF-AA construct. cIAP1-B3R binds ubiquitin with a KD of approximately 20 μM as determined by a single-site binding isotherm fit, in good agreement with past studies2. Recent reports have suggested that this mutation destabilizes cIAP13, which might contribute to the very slight decrease in E2 binding affinity we observe in the MF-AA variants. The use of cIAP1MF-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-ΔCARD (green) to E2SRCK 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 μM (cIAP1-B3R) and 19 μM (cIAP1-B3R-ΔCARD), recapitulating the relative increase in affinity upon CARD deletion observed in the case of cIAP1-B3RMF-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 μM). 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 μM) 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 μM) was incubated with UbcH5c at 0 to 128 μM. No detectable dimer is formed. cIAP1 bound to BV6 is included as a dimerization control.

Supplementary Figure 3 Methionine methyl groups assigned by mutagenesis.

1H-13C 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 kex = 970 ± 100 s-1 and pB = 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 R2 dispersion, Q411 and L418, are severely overlapped in the 1H-15N spectra. As shown in Fig. 4a, R2 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-ΔCARD (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-B3RL617E 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-ΔC7 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(θ)/λ, where 2θ = the angle between the incident X-ray beam and the detector, and λ = the X-ray wavelength in Ångstroms). (b) The same data as in (a) is shown for cIAP1-B3R-ΔC7 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 data1. (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 Rg values in the ensembles of molecular models generated by EOM for cIAP1-B3R (black), cIAP1-B3RL617E (orange), cIAP1-B3R-ΔC7 (violet) and cIAP1-B3RL617E + 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-B3RL617E) adopt tighter and smaller distributions than the open states (cIAP1-B3R-ΔC7 and cIAP1-B3RL617E + 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-B3RL617E 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-B3RL617E 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.

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Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Tables 1–3 (PDF 1897 kb)

Supplementary Data Set 1

Uncropped gels from Figure 1d,e and Supplementary Figure 2 (PDF 15563 kb)

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Phillips, A., Schoeffler, A., Matsui, T. et al. Internal motions prime cIAP1 for rapid activation. Nat Struct Mol Biol 21, 1068–1074 (2014). https://doi.org/10.1038/nsmb.2916

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