Mechanism of ubiquitin ligation and lysine prioritization by a HECT E3.
Ubiquitination by HECT E3 enzymes regulates myriad processes, including tumor suppression, transcription, protein trafficking, and degradation. HECT E3s use a two-step mechanism to ligate ubiquitin to target proteins. The first step is guided by interactions between the catalytic HECT domain and the E2∼ubiquitin intermediate, which promote formation of a transient, thioester-bonded HECT∼ubiquitin intermediate. Here we report that the second step of ligation is mediated by a distinct catalytic architecture established by both the HECT E3 and its covalently linked ubiquitin. The structure of a chemically trapped proxy for an E3∼ubiquitin-substrate intermediate reveals three-way interactions between ubiquitin and the bilobal HECT domain orienting the E3∼ubiquitin thioester bond for ligation, and restricting the location of the substrate-binding domain to prioritize target lysines for ubiquitination. The data allow visualization of an E2-to-E3-to-substrate ubiquitin transfer cascade, and show how HECT-specific ubiquitin interactions driving multiple reactions are repurposed by a major E3 conformational change to promote ligation. DOI:http://dx.doi.org/10.7554/eLife.00828.001.
Department of Structural Biology , St Jude Children's Research Hospital , Memphis , United States.
Hari B
HB
Kamadurai
Yu
Y
Qiu
Alan
A
Deng
Joseph S
JS
Harrison
Chris
C
Macdonald
Marcelo
M
Actis
Patrick
P
Rodrigues
Darcie J
DJ
Miller
Judith
J
Souphron
Steven M
SM
Lewis
Igor
I
Kurinov
Naoaki
N
Fujii
Michal
M
Hammel
Robert
R
Piper
Brian
B
Kuhlman
Brenda A
BA
Schulman
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; D008239:Lysine
E2 conjugating enzyme; E3 ligase; HECT; NEDD4; Rsp5; S. cerevisiae; ubiquitin