Article | Published:

Internal motions prime cIAP1 for rapid activation

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

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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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.

Author information

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.

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.

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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    Supplementary Data Set 1

    Uncropped gels from Figure 1d,e and Supplementary Figure 2

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DOI

https://doi.org/10.1038/nsmb.2916

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