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Biochemical and Structural Characterization of the Ubiquitin-Conjugating Enzyme UBE2W Reveals the Formation of a Noncovalent Homodimer

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Abstract

The biochemical and structural characterization of ubiquitin-conjugating enzymes (E2s) over the past 30 years has fostered important insights into ubiquitin transfer mechanisms. Although many of these enzymes share high sequence and structural conservation, their functional roles in the cell are decidedly diverse. Here, we report that the mono-ubiquitinating E2 UBE2W forms a homodimer using two distinct protein surfaces. Dimerization is primarily driven by residues in the ß-sheet region and Loops 4 and 7 of the catalytic domain. Mutation of two residues in the catalytic domain of UBE2W is capable of disrupting UBE2W homodimer formation, however, we find that dimerization of this E2 is not required for its ubiquitin transfer activity. In addition, residues in the C-terminal region, although not compulsory for the dimerization of UBE2W, play an ancillary role in the dimer interface. In all current E2 structures, the C-terminal helix of the UBC domain is at least 15Å away from the primary dimerization surface shown here for UBE2W. This leads to the proposal that the C-terminal region of UBE2W adopts a noncanonical position that places it closer to the UBC ß-sheet, providing the first indication that at least some E2s adopt C-terminal conformations different from the canonical structures observed to date.

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Notes

  1. E2s are organized into classes: Class I E2s contain only the ~150-residue catalytic domain (“UBC” domain); Class II, III, and IV E2s also contain N-terminal, C-terminal extension, or both. Currently, little is known regarding the function of such extensions and there is little structural information available on any extensions. All available E2 structures, regardless of the class they belong to, are of the UBC domain.

  2. Of the ~120 resonances in the UBE2W-‘KK’ 1H, 15N-HSQC spectrum, we were able to unambiguously assign 115. From this dataset, we were able to transfer assignments to the ‘WT’ spectrum and identify 80 of the ~90 resonances present.

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Acknowledgments

The authors acknowledge D. Christensen and C. Eakin for their initial observations on the UBE2W-KK mutant. This study was supported by the National Institute of General Medical Sciences Grants R01 GM088055 (R.E.K.). V. V. was supported in part by the Hurd Fellowship Fund and PHS NRSA 2T32 GM007270.

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Authors and Affiliations

  1. Department of Biochemistry, University of Washington, Seattle, WA, 98195, USA

    Vinayak Vittal, Dawn M. Wenzel, Peter S. Brzovic & Rachel E. Klevit

  2. Department of Biochemistry, University of Utah, Salt Lake City, UT, 84112, USA

    Dawn M. Wenzel

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  1. Vinayak Vittal
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  2. Dawn M. Wenzel
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Correspondence to Rachel E. Klevit.

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Vittal, V., Wenzel, D.M., Brzovic, P.S. et al. Biochemical and Structural Characterization of the Ubiquitin-Conjugating Enzyme UBE2W Reveals the Formation of a Noncovalent Homodimer. Cell Biochem Biophys 67, 103–110 (2013). https://doi.org/10.1007/s12013-013-9633-5

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