Premium
Unraveling the Activation Mechanism of Cullin‐RING Ubiquitin Ligases using Quantitative Cross‐linking Mass Spectrometry
Author(s) -
Yu Clinton,
Mao Haibin,
Novitsky Eric,
Rychnovsky Scott D.,
Zheng Ning,
Huang Lan
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.595.6
Subject(s) - cullin , nedd8 , neddylation , ubiquitin , biochemistry , chemistry , ubiquitin ligase , microbiology and biotechnology , protein–protein interaction , protein structure , biology , gene
Cullin‐RING ubiquitin ligases (CRLs) represent a superfamily of heterogeneous multi‐protein E3 ubiquitin ligases comprised of a cullin and RING protein catalytic core that mediate the ubiquitination of protein substrates. The full enzymatic activity of CRLs requires cullin modification by Nedd8 (i.e. neddylation). This ubiquitin‐like modification promotes both E2 recruitment and subsequent ubiquitin transfer, thereby stimulating the E3 activity of CRL ligases. Thus, neddylation represents a critical post‐translational modification that regulates protein ubiquitination by these complexes. Despite extensive structural studies on CRLs, certain structural details of CRLs during protein ubiquitination remain elusive. It has been shown that deamidation of Gln40 of Nedd8 to glutamate (Q40E) effectively inhibits CRL E3 activities, directly interfering with cellular functions. However, the molecular mechanism by which Nedd8(Q40E) affects CRL function is unclear. Cross‐linking mass spectrometry (XL‐MS) is a powerful strategy that can be utilized to characterize dynamic and heterogeneous protein complexes that are difficult to study using conventional structural tools. Quantitative cross‐linking mass spectrometry (QXL‐MS) not only allows the determination of physical contacts between and within proteins at the amino acid resolution, but also enables quantitative assessment of conformational changes in protein complexes in various states. To elucidate the structural basis underlying the activation/inactivation of CRLs through neddylation, we have employed novel QXL‐MS strategies to compare cross‐link profiles of different types of full‐length human Cul1‐Rbx1 complexes, as well as their interactions with E2‐conjugating enzyme Cdc34. Thus far we have uncovered major Nedd8‐induced structural rearrangements of the CRL1 catalytic core that are not induced by Nedd8(Q40E) conjugation, suggesting that the subtle change of a single Nedd8 amino acid is sufficient to re‐orient the topology of the CRL catalytic core. Our results provide new insights into how neddylation regulates the conformation and activity of CRLs. Support or Funding Information This work is supported by NIH RO1GM074830 to L.H, and R01GM106003 to L.H. and S. R..