A dual E3 mechanism for Rub1 ligation to Cdc53.
In ubiquitin-like protein (UBL) cascades, a thioester-linked E2∼UBL complex typically interacts with an E3 enzyme for UBL transfer to the target. Here we demonstrate a variant mechanism, whereby the E2 Ubc12 functions with two E3s, Hrt1 and Dcn1, for ligation of the UBL Rub1 to Cdc53's WHB subdomain. Hrt1 functions like a conventional RING E3, with its N terminus recruiting Cdc53 and C-terminal RING activating Ubc12∼Rub1. Dcn1's "potentiating neddylation" domain (Dcn1(P)) acts as an additional E3, reducing nonspecific Hrt1-mediated Ubc12∼Rub1 discharge and directing Ubc12's active site to Cdc53. Crystal structures of Dcn1(P)-Cdc53(WHB) and Ubc12 allow modeling of a catalytic complex, supported by mutational data. We propose that Dcn1's interactions with both Cdc53 and Ubc12 would restrict the otherwise flexible Hrt1 RING-bound Ubc12∼Rub1 to a catalytically competent orientation. Our data reveal mechanisms by which two E3s function synergistically to promote UBL transfer from one E2 to a target.
Scott
Daniel C
DC
Howard Hughes Medical Institute, St Jude Children's Research Hospital, Memphis, TN 38105, USA.
Monda
Julie K
JK
Grace
Christy R R
CR
Duda
David M
DM
Kriwacki
Richard W
RW
Kurz
Thimo
T
Schulman
Brenda A
BA
D018360:Crystallography, X-Ray; D044842:Cullin Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D044843:SKP Cullin F-Box Protein Ligases / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D014452:Ubiquitins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
C100988:Cdc53 protein, S cerevisiae; D044842:Cullin Proteins; C502135:Dcn1 protein, S cerevisiae; D009097:Multienzyme Complexes; C111706:RUB1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C112595:UBC12 protein, S cerevisiae; D014452:Ubiquitins; C485632:HRT1 protein, S cerevisiae; D044843:SKP Cullin F-Box Protein Ligases; D044767:Ubiquitin-Protein Ligases