Article | Published:

Internal motions prime cIAP1 for rapid activation

Nature Structural & Molecular Biology volume 21, pages 10681074 (2014) | Download Citation


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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  1. 1.

    & Hallmarks of cancer: the next generation. Cell 144, 646–674 (2011).

  2. 2.

    & Programmed cell death in animal development and disease. Cell 147, 742–758 (2011).

  3. 3.

    & Small-molecule pan-IAP antagonists: a patent review. Expert Opin. Ther. Pat. 20, 251–267 (2010).

  4. 4.

    & Targeting IAP proteins for therapeutic intervention in cancer. Nat. Rev. Drug Discov. 11, 109–124 (2012).

  5. 5.

    , , , & Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell 102, 33–42 (2000).

  6. 6.

    et al. Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell 102, 43–53 (2000).

  7. 7.

    et al. Structural basis for binding of Smac/DIABLO to the XIAP BIR3 domain. Nature 408, 1004–1008 (2000).

  8. 8.

    et al. Structural basis of IAP recognition by Smac/DIABLO. Nature 408, 1008–1012 (2000).

  9. 9.

    et al. Engineering ML-IAP to produce an extraordinarily potent caspase 9 inhibitor: implications for Smac-dependent anti-apoptotic activity of ML-IAP. Biochem. J. 385, 11–20 (2005).

  10. 10.

    et al. Design, synthesis, and biological activity of a potent Smac mimetic that sensitizes cancer cells to apoptosis by antagonizing IAPs. ACS Chem. Biol. 1, 525–533 (2006).

  11. 11.

    et al. IAP antagonists induce autoubiquitination of c-IAPs, NF-κB activation, and TNFα-dependent apoptosis. Cell 131, 669–681 (2007).

  12. 12.

    et al. A Smac mimetic rescue screen reveals roles for inhibitor of apoptosis proteins in tumor necrosis factor-α signaling. Cancer Res. 67, 11493–11498 (2007).

  13. 13.

    et al. IAP antagonists target cIAP1 to induce TNFα-dependent apoptosis. Cell 131, 682–693 (2007).

  14. 14.

    et al. cIAP1 and cIAP2 facilitate cancer cell survival by functioning as E3 ligases that promote RIP1 ubiquitination. Mol. Cell 30, 689–700 (2008).

  15. 15.

    et al. c-IAP1 and c-IAP2 are critical mediators of tumor necrosis factor alpha (TNFα)-induced NF-κB activation. J. Biol. Chem. 283, 24295–24299 (2008).

  16. 16.

    , , , & Structure of a RING E3 ligase and ubiquitin-loaded E2 primed for catalysis. Nature 489, 115–120 (2012).

  17. 17.

    , , , & BIRC7–E2 ubiquitin conjugate structure reveals the mechanism of ubiquitin transfer by a RING dimer. Nat. Struct. Mol. Biol. 19, 876–883 (2012).

  18. 18.

    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).

  19. 19.

    et al. Smac mimetics activate the E3 ligase activity of cIAP1 protein by promoting RING domain dimerization. J. Biol. Chem. 286, 17015–17028 (2011).

  20. 20.

    et al. Antagonists induce a conformational change in cIAP1 that promotes autoubiquitination. Science 334, 376–380 (2011).

  21. 21.

    et al. CARD-mediated autoinhibition of cIAP1′s E3 ligase activity suppresses cell proliferation and migration. Mol. Cell 42, 569–583 (2011).

  22. 22.

    & An introduction to NMR-based approaches for measuring protein dynamics. Biochim. Biophys. Acta 1814, 942–968 (2011).

  23. 23.

    & Methyl groups as probes of structure and dynamics in NMR studies of high-molecular-weight proteins. ChemBioChem 6, 1567–1577 (2005).

  24. 24.

    et al. Structural basis for signal-sequence recognition by the translocase motor SecA as determined by NMR. Cell 131, 756–769 (2007).

  25. 25.

    , , , & An integrated high-throughput data acquisition system for biological solution X-ray scattering studies. J. Synchrotron Radiat. 19, 431–434 (2012).

  26. 26.

    , , , & Structural characterization of flexible proteins using small-angle X-ray scattering. J. Am. Chem. Soc. 129, 5656–5664 (2007).

  27. 27.

    , , , & PRIMUS: a Windows PC-based system for small-angle scattering data analysis. J. Appl. Crystallogr. 36, 1277–1282 (2003).

  28. 28.

    & Dynamic personalities of proteins. Nature 450, 964–972 (2007).

  29. 29.

    , , & Macromolecular juggling by ubiquitylation enzymes. BMC Biol. 11, 65 (2013).

  30. 30.

    , & Kinetics of the low pH-induced conformational changes and fusogenic activity of influenza hemagglutinin. Biophys. J. 67, 2355–2360 (1994).

  31. 31.

    & A spring-loaded mechanism for the conformational change of influenza hemagglutinin. Cell 73, 823–832 (1993).

  32. 32.

    et al. Structure of the UBA domain of Dsk2p in complex with ubiquitin molecular determinants for ubiquitin recognition. Structure 13, 521–532 (2005).

  33. 33.

    , , , & UbcH5/ubiquitin noncovalent complex is required for processive BRCA1-directed ubiquitination. Mol. Cell 21, 873–880 (2006).

  34. 34.

    et al. Structure of an E3:E2~Ub complex reveals an allosteric mechanism shared among RING/U-box ligases. Mol. Cell 47, 933–942 (2012).

  35. 35.

    et al. Recognition of UbcH5c and the nucleosome by the Bmi1/Ring1b ubiquitin ligase complex. EMBO J. 30, 3285–3297 (2011).

  36. 36.

    et al. Preparation of distinct ubiquitin chain reagents of high purity and yield. Structure 19, 1053–1063 (2011).

  37. 37.

    et al. A plasmid expression system for quantitative in vivo biotinylation of thioredoxin fusion proteins in Escherichia coli. Nucleic Acids Res. 26, 1414–1420 (1998).

  38. 38.

    et al. An efficient and cost-effective isotope labeling protocol for proteins expressed in Escherichia coli. J. Biomol. NMR 11, 97–102 (1998).

  39. 39.

    Isotope labeling strategies for the study of high-molecular-weight proteins by solution NMR spectroscopy. Nat. Protoc. 1, 749–754 (2006).

  40. 40.

    , , , & Slow dynamics in folded and unfolded states of an SH3 domain. J. Am. Chem. Soc. 123, 11341–11352 (2001).

  41. 41.

    & E. A general two-site solution for the chemical exchange produced dependence of T2 upon the Carr-Purcell pulse separation. J. Magn. Reson. 6, 89–105 (1972).

  42. 42.

    & GUARDD: user-friendly MATLAB software for rigorous analysis of CPMG RD NMR data. J. Biomol. NMR 52, 11–22 (2012).

  43. 43.

    et al. A wide-bandpass multilayer monochromator for biological small-angle scattering and fiber diffraction studies. J. Appl. Crystallogr. 31, 672–682 (1998).

  44. 44.

    Determination of the regularization parameter in indirect-transform methods using perceptual criteria. J. Appl. Crystallogr. 25, 495–503 (1992).

  45. 45.

    , & Determination of domain structure of proteins from X-ray solution scattering. Biophys. J. 80, 2946–2953 (2001).

  46. 46.

    & Uniqueness of ab initio shape determination in small-angle scattering. J. Appl. Crystallogr. 36, 860–864 (2003).

  47. 47.

    & Global rigid body modeling of macromolecular complexes against small-angle scattering data. Biophys. J. 89, 1237–1250 (2005).

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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

    • Aaron H Phillips
    •  & John W Blankenship

    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.).

    • Aaron H Phillips
    •  & Allyn J Schoeffler

    These authors contributed equally to this work.


  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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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.

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.

Corresponding authors

Correspondence to Erin C Dueber or Wayne J Fairbrother.

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