Journal of Molecular Biology
Volume 392, Issue 5, 9 October 2009, Pages 1117-1124
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The Structure and Conformation of Lys63-Linked Tetraubiquitin

https://doi.org/10.1016/j.jmb.2009.07.090Get rights and content

Summary

Ubiquitination involves the covalent attachment of the ubiquitin (Ub) C-terminus to the lysine side chain of a substrate protein by an isopeptide bond. The modification can comprise a single Ub moiety or a chain of Ub molecules joined by isopeptide bonds between the C-terminus of one Ub with one of the seven lysine residues in the next Ub. Modification of substrate proteins with Lys63-linked poly-Ub plays a key nondegradative signaling role in many biological processes, including DNA repair and nuclear factor-κB activation, whereas substrates modified by Lys48-linked chains are targeted to the proteasome for degradation. The distinct signaling properties of alternatively linked Ub chains presumably stem from structural differences that can be distinguished by effector proteins. We have determined the crystal structure of Lys63 tetra-Ub at a resolution of 1.96 Å and performed small-angle X-ray scattering experiments and molecular dynamics simulations to probe the conformation of Lys63 tetra-Ub in solution. The chain adopts a highly extended conformation in the crystal, in contrast with the compact globular fold of Lys48 tetra-Ub. Small-angle X-ray scattering experiments show that the Lys63 tetra-Ub chain is dynamic in solution, adopting an ensemble of conformations that are more compact than the extended form in the crystal. The results of these studies provide a basis for understanding the differences in the behavior and recognition of Lys63 poly-Ub chains.

Section snippets

Experimental details

Lys63-linked tetra-Ub chain was synthesized using two Ub mutants (K48R–K63R and Ub-D77) following Pickart and Raasi.23 All the enzymes required for this synthesis, viz., E1 (human), Ubc13/MMS2 (yeast), and Yuh1 (yeast), were also expressed and purified as recombinant proteins in Escherichia coli. After the chain of required length was synthesized, it was subjected to a final step of purification using size-exclusion chromatography in Superdex 75 prep-grade 26/60 column. The protein was then

Accession number

Coordinates and structure factors have been deposited in the Protein Data Bank with accession number 3HM3.

Acknowledgements

This work was supported by the Howard Hughes Medical Institute (A.B.D and C.W.) and the National Cancer Institute (SBDR grant no. CA92584; G.H.). We thank Michal Hammel from the Lawrence Berkeley National Laboratory for helping with the implementation of MES.

References (41)

  • Vijay-KumarS. et al.

    Structure of ubiquitin refined at 1.8 Å resolution

    J. Mol. Biol.

    (1987)
  • RaasiS. et al.

    Binding of polyubiquitin chains to ubiquitin-associated (UBA) domains of HHR23A

    J. Mol. Biol.

    (2004)
  • EaC. et al.

    Activation of IKK by TNFalpha requires site-specific ubiquitination of RIP1 and polyubiquitin binding by NEMO

    Mol. Cell

    (2006)
  • RahighiS. et al.

    Specific recognition of linear ubiquitin chains by NEMO is important for NF-kappaB activation

    Cell

    (2009)
  • HaglundK. et al.

    Ubiquitylation and cell signaling

    EMBO J.

    (2005)
  • MukhopadhyayD. et al.

    Proteasome-independent functions of ubiquitin in endocytosis and signaling

    Science

    (2007)
  • HershkoA. et al.

    The ubiquitin system

    Annu. Rev. Biochem.

    (1998)
  • KirisakoT. et al.

    A ubiquitin ligase complex assembles linear polyubiquitin chains

    EMBO J.

    (2006)
  • PickartC.M. et al.

    Proteasomes and their kin: proteases in the machine age

    Nat. Rev. Mol. Cell Biol.

    (2004)
  • ChenZ.J.

    Ubiquitin signalling in the NF-kappaB pathway

    Nat. Cell Biol.

    (2005)
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