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UBCH7 reactivity profile reveals parkin and HHARI to be RING/HECT hybrids

Nature volume 474, pages 105–108 (2011)Cite this article

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Abstract

Although the functional interaction between ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s) is essential in ubiquitin (Ub) signalling, the criteria that define an active E2–E3 pair are not well established. The human E2 UBCH7 (also known as UBE2L3) shows broad specificity for HECT-type E3s1, but often fails to function with RING E3s in vitro despite forming specific complexes2,3,4. Structural comparisons of inactive UBCH7–RING complexes with active UBCH5–RING complexes reveal no defining differences3,4, highlighting a gap in our understanding of Ub transfer. Here we show that, unlike many E2s that transfer Ub with RINGs, UBCH7 lacks intrinsic, E3-independent reactivity with lysine, explaining its preference for HECTs. Despite lacking lysine reactivity, UBCH7 exhibits activity with the RING-in-between-RING (RBR) family of E3s that includes parkin (also known as PARK2) and human homologue of ariadne (HHARI; also known as ARIH1)5,6. Found in all eukaryotes7, RBRs regulate processes such as translation8 and immune signalling9. RBRs contain a canonical C3HC4-type RING, followed by two conserved Cys/His-rich Zn2+-binding domains, in-between-RING (IBR) and RING2 domains, which together define this E3 family7. We show that RBRs function like RING/HECT hybrids: they bind E2s via a RING domain, but transfer Ub through an obligate thioester-linked Ub (denoted ∼Ub), requiring a conserved cysteine residue in RING2. Our results define the functional cadre of E3s for UBCH7, an E2 involved in cell proliferation10 and immune function11, and indicate a novel mechanism for an entire class of E3s.

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Figure 1: UBCH7 does not react with free lysine.
Figure 2: Lysine reactivity is multifactorial.
Figure 3: RBR E3s function via a HECT-like mechanism.
Figure 4: Cysteine C357 is the active site of HHARI.

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Acknowledgements

We thank K. Stoll, M. Schwartz and N. Zheng for critical reading of the manuscript and discussions and V. Vittal, K. Stoll and P. Littlefield for technical help. We thank K. Borden, E. Fon, B. Schulman, L. Boyd, J. Chen and A. Merz for expression vectors for HHARI, parkin, E6-AP, Ubc18, human E1 and GST pull-down reagents, respectively. We acknowledge support from the National Institute of General Medical Sciences in the form of 5R01 GM088055 (R.E.K.) and T32 GM07270 (D.M.W.), and the National Science Foundation in the form of MCB0615632 (R.E.K.)

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  1. Department of Biochemistry, Box 357350, University of Washington, Seattle, 98195, Washington, USA

    Dawn M. Wenzel, Alexei Lissounov, Peter S. Brzovic & Rachel E. Klevit

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  1. Dawn M. Wenzel
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Contributions

D.M.W. performed all experiments. P.S.B. and A.L. contributed to the experimental design for Fig. 2 and Supplementary Fig. 5. D.M.W. and R.E.K. designed the overall study and wrote the manuscript with P.S.B.

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Correspondence to Rachel E. Klevit.

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The authors declare no competing financial interests.

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Wenzel, D., Lissounov, A., Brzovic, P. et al. UBCH7 reactivity profile reveals parkin and HHARI to be RING/HECT hybrids. Nature 474, 105–108 (2011). https://doi.org/10.1038/nature09966

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