The HIP2~Ubiquitin Conjugate Forms a Non-Compact Monomeric Thioester during Di-Ubiquitin Synthesis

Cook, Benjamin W.; Barber, Kathryn R.; Shilton, Brian H.; Shaw, Gary S.; Massiah, Michael

doi:10.1371/journal.pone.0120318

The HIP2~Ubiquitin Conjugate Forms a Non-Compact Monomeric Thioester during Di-Ubiquitin Synthesis

Journal Article · Mon Mar 23 00:00:00 EDT 2015 · PLoS ONE

DOI:https://doi.org/10.1371/journal.pone.0120318· OSTI ID:1404952

Cook, Benjamin W. ^[1]; Barber, Kathryn R. ^[1]; Shilton, Brian H. ^[1]; Shaw, Gary S. ^[1]; Massiah, Michael ^[2]

Univ. of Western Ontario, London, ON (Canada)
George Washington Univ., Washington, DC (United States)

Polyubiquitination is a post-translational event used to control the degradation of damaged or unwanted proteins by modifying the target protein with a chain of ubiquitin molecules. One potential mechanism for the assembly of polyubiquitin chains involves the dimerization of an E2 conjugating enzyme allowing conjugated ubiquitin molecules to be put into close proximity to assist reactivity. HIP2 (UBE2K) and Ubc1 (yeast homolog of UBE2K) are unique E2 conjugating enzymes that each contain a C-terminal UBA domain attached to their catalytic domains, and they have basal E3-independent polyubiquitination activity. Although the isolated enzymes are monomeric, polyubiquitin formation activity assays show that both can act as ubiquitin donors or ubiquitin acceptors when in the activated thioester conjugate suggesting dimerization of the E2-ubiquitin conjugates. Stable disulfide complexes, analytical ultracentrifugation and small angle x-ray scattering were used to show that the HIP2-Ub and Ubc1-Ub thioester complexes remain predominantly monomeric in solution. Models of the HIP2-Ub complex derived from SAXS data show the complex is not compact but instead forms an open or backbent conformation similar to UbcH5b~Ub or Ubc13~Ub where the UBA domain and covalently attached ubiquitin reside on opposite ends of the catalytic domain. Activity assays showed that full length HIP2 exhibited a five-fold increase in the formation rate of di-ubiquitin compared to a HIP2 lacking the UBA domain. This difference was not observed for Ubc1 and may be attributed to the closer proximity of the UBA domain in HIP2 to the catalytic core than for Ubc1.

View Accepted Manuscript (DOE)

Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States)

Sponsoring Organization:: Canadian Inst. of Health; National Inst. of Health Research; USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)

Grant/Contract Number:: W-31109-ENG-38

OSTI ID:: 1404952

Journal Information:: PLoS ONE, Journal Name: PLoS ONE Journal Issue: 3 Vol. 10; ISSN 1932-6203

Publisher:: Public Library of ScienceCopyright Statement

Country of Publication:: United States

Language:: ENGLISH

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The molecular basis of lysine 48 ubiquitin chain synthesis by Ube2K Middleton, Adam J.; Day, Catherine L. Scientific Reports, Vol. 5, Issue 1 https://doi.org/10.1038/srep16793	journal	November 2015

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The HIP2~Ubiquitin Conjugate Forms a Non-Compact Monomeric Thioester during Di-Ubiquitin Synthesis

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