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Title: A UbcH5/Ubiquitin Noncovalent Complex is Required for Processive BRCA1-Directed Ubiquitination

Abstract

Protein ubiquitination is a powerful regulatory modification that influences nearly every aspect of eukaryotic cell biology. The general pathway for ubiquitin (Ub) modification requires the sequential activities of a Ub-activating enzyme (E1), a Ub transfer enzyme (E2), and a Ub ligase (E3). The E2 must recognize both the E1 and a cognate E3 in addition to carrying activated Ub. These central functions are performed by a topologically conserved a/b-fold core domain ofw150 residues shared by all E2s. However, as presented herein, the UbcH5 family of E2s can also bind Ub noncovalently on a surface well removed from the E2 active site. We present the solution structure of the UbcH5c/ Ub noncovalent complex and demonstrate that this noncovalent interaction permits self-assembly of activated UbcH5cwUb molecules. Self-assembly has profound consequences for the processive formation of polyubiquitin (poly-Ub) chains in ubiquitination reactions directed by the breast and ovarian cancer tumor susceptibility protein BRCA1

Authors:
; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
Sponsoring Org.:
USDOE
OSTI Identifier:
881096
Report Number(s):
PNNL-SA-48810
2612d; 2447a; 2034a; 2612c; 2612b; 2612a; KP1704020; TRN: US200623%%609
DOE Contract Number:
AC05-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: Molecular Cell, 21(6):873-880
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; BIOLOGY; ENZYMES; LIGASES; MAMMARY GLANDS; MODIFICATIONS; NEOPLASMS; PROTEINS; RESIDUES; Environmental Molecular Sciences Laboratory

Citation Formats

Brzovic, Peter S., Lissounov, Alexei V., Christensen, Devin, Hoyt, David W., and Klevit, Rachel E.. A UbcH5/Ubiquitin Noncovalent Complex is Required for Processive BRCA1-Directed Ubiquitination. United States: N. p., 2006. Web. doi:10.1016/j.molcel.2006.02.008.
Brzovic, Peter S., Lissounov, Alexei V., Christensen, Devin, Hoyt, David W., & Klevit, Rachel E.. A UbcH5/Ubiquitin Noncovalent Complex is Required for Processive BRCA1-Directed Ubiquitination. United States. doi:10.1016/j.molcel.2006.02.008.
Brzovic, Peter S., Lissounov, Alexei V., Christensen, Devin, Hoyt, David W., and Klevit, Rachel E.. Fri . "A UbcH5/Ubiquitin Noncovalent Complex is Required for Processive BRCA1-Directed Ubiquitination". United States. doi:10.1016/j.molcel.2006.02.008.
@article{osti_881096,
title = {A UbcH5/Ubiquitin Noncovalent Complex is Required for Processive BRCA1-Directed Ubiquitination},
author = {Brzovic, Peter S. and Lissounov, Alexei V. and Christensen, Devin and Hoyt, David W. and Klevit, Rachel E.},
abstractNote = {Protein ubiquitination is a powerful regulatory modification that influences nearly every aspect of eukaryotic cell biology. The general pathway for ubiquitin (Ub) modification requires the sequential activities of a Ub-activating enzyme (E1), a Ub transfer enzyme (E2), and a Ub ligase (E3). The E2 must recognize both the E1 and a cognate E3 in addition to carrying activated Ub. These central functions are performed by a topologically conserved a/b-fold core domain ofw150 residues shared by all E2s. However, as presented herein, the UbcH5 family of E2s can also bind Ub noncovalently on a surface well removed from the E2 active site. We present the solution structure of the UbcH5c/ Ub noncovalent complex and demonstrate that this noncovalent interaction permits self-assembly of activated UbcH5cwUb molecules. Self-assembly has profound consequences for the processive formation of polyubiquitin (poly-Ub) chains in ubiquitination reactions directed by the breast and ovarian cancer tumor susceptibility protein BRCA1},
doi = {10.1016/j.molcel.2006.02.008},
journal = {Molecular Cell, 21(6):873-880},
number = ,
volume = ,
place = {United States},
year = {Fri Mar 17 00:00:00 EST 2006},
month = {Fri Mar 17 00:00:00 EST 2006}
}
  • BRCA1 is a breast and ovarian cancer tumor suppressor protein that associates with BARD1 to form a RING/RING heterodimer. The BRCA1/BARD1 RING complex functions as an ubiquitin (Ub) ligase with activity substantially greater than individual BRCA1 or BARD1 subunits. By using NMR spectroscopy and site-directed mutagenesis, we have mapped the binding site on the BRCA1/BARD1 heterodimer for the Ub-conjugating enzyme UbcH5c. The results demonstrate that UbcH5c binds only to the BRCA1 RING domain and not the BARD1 RING. The binding interface is formed by the first and second Zn2+-loops and central -helix of the BRCA1 RING domain, a region disruptedmore » by cancer-predisposing mutations. Unexpectedly, a second Ub-conjugating enzyme, UbcH7, also interacts with the BRCA1/BARD1 complex with similar affinity, although it is not active in Ub-ligase activity assays. Thus, binding alone is not sufficient for BRCA1-dependent Ub-ligase activity.« less
  • The F-box proteins are the substrate recognition subunits of the SCF (Skp1-Cul1-Rbx1-F-box protein) ubiquitin ligase complexes that control the stability of numerous regulators in eukaryotic cells. Here we show that dimerization of the F-box protein Fbx4 is essential for SCF{sup Fbx4} (the superscript denotes the F-box protein) ubiquitination activity toward the telomere regulator Pin2 (also known as TRF1). The crystal structure of Fbx4 in complex with an adaptor protein Skp1 reveals an antiparallel dimer configuration in which the linker domain of Fbx4 interacts with the C-terminal substrate-binding domain of the other protomer, whereas the C-terminal domain of the protein adoptsmore » a compact {alpha}/{beta} fold distinct from those of known F-box proteins. Biochemical studies indicate that both the N-terminal domain and a loop connecting the linker and C-terminal domain of Fbx4 are critical for the dimerization and activation of the protein. Our findings provide a framework for understanding the role of F-box dimerization in the SCF-mediated ubiquitination reaction.« less
  • We characterize a novel microsome system that forms high-molecular-mass (HMM) CYP3A, CYP2E1, and ubiquitin conjugates, but does not alter CYP4A or most other microsomal proteins. The formation of the HMM bands was observed in hepatic microsomes isolated from rats treated 1 week or more with high doses (50 mg/kg/day) of nicardipine, clotrimazole, or pregnenolone 16alpha-carbonitrile, but not microsomes from control, dexamethasone-, nifedipine-, or diltiazem-treated rats. Extensive washing of the microsomes to remove loosely attached proteins or cytosolic contaminants did not prevent the conjugation reaction. In contrast to prototypical ubiquitination pathways, this reaction did not require addition of ubiquitin, ATP, Mg(2+),more » or cytosol. Addition of cytosol did result in the degradation of the HMM CYP3A bands in a process that was not blocked by proteasome inhibitors. Immunoprecipitated CYP3A contained HMM ubiquitin. Even so, mass spectrometric analysis of tryptic peptides indicated that the HMM CYP3A was in molar excess to ubiquitin, suggesting that the formation of the HMM CYP3A may have resulted from conjugation to itself or a diffuse pool of ubiquitinated proteins already present in the microsomes. Addition of CYP3A substrates inhibited the formation of the HMM CYP3A and the cytosol-dependent degradation of HMM CYP3A. These results suggest that after extended periods of elevated CYP3A expression, microsomal factors are induced that catalyze the formation of HMM CYP3A conjugates that contain ubiquitin. This conjugation reaction, however, seems to be distinct from the classical ubiquitination pathway but may be related to the substrate-dependent stabilization of CYP3A observed in vivo.« less