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. 2010 Sep 10;39(5):784-96.
doi: 10.1016/j.molcel.2010.08.030.

A dual E3 mechanism for Rub1 ligation to Cdc53

Daniel C Scott  1 , Julie K Monda, Christy R R Grace, David M Duda, Richard W Kriwacki, Thimo Kurz, Brenda A Schulman
Affiliations

A dual E3 mechanism for Rub1 ligation to Cdc53

Daniel C Scott et al. Mol Cell. .

Abstract

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.

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Figures

Figure 1
Figure 1. Hrt1 RING-type E3 activity for Rub1 modification of Cdc53
Error bars - +/− 1 standard deviation from 3 independent experiments. A. Time course of Ubc12~Rub1 discharge to hydroxylamine. Ubc12 was loaded with [32P]-Rub1 by Ula1-Uba3 in the presence of MgATP. The reaction was treated with EDTA to prevent subsequent [32P]-Rub1 loading of Ubc12, and hydroxylamine was added in the presence or absence of Hrt1RING or Dcn1P. Aliquots were removed at the indicated times and added to nonreducing SDS sample buffer prior to analysis by SDS-PAGE/Phosphorimager. B. Time course of Ubc12~[32P]-Rub1 discharge to hydroxylamine in the presence or absence of Cdc53C+/K760R-Hrt1 or Cdc53C+-Hrt1ΔR. Note different time-scale from (A). C. Phosphorimager data for time-course of pulse-chase assay monitoring [32P]-Rub1 transfer from Ubc12 to Cdc53C+ with the indicated versions of Hrt1, and the isolated Hrt1RING as indicated. D. Crystal structure of S. cerevisiae Ubc12. The two molecules in the asymmetric unit (Chain A cyan; Chain B grey) are shown superimposed with the prior structure of the catalytic core domain of human Ubc12 (red) (Huang et al., 2005). E. Same as (B), but comparing the function of the Ubc12 core domain lacking the N-terminal helix (Ubc12core), and with quantification. Standard error from 3 independent experiments is shown. F. Structural model of the yeast Ubc12 core domain (cyan, residues 25 to the C-terminal 188) associated with the Hrt1 RING (blue, with zinc atoms as grey spheres, left) based on UbcH7 (cyan)-cCbl RING (blue, with zinc atoms as grey spheres, right) (Zheng et al., 2000). G. Same as (A), but with the indicated versions of Ubc12 mutated at the predicted interface with the Hrt1 RING, and with quantification included for comparison between Ubc12 and mutants. Note different time-scale from (E). H. Phosphorimager data for time-course of pulse-chase assay monitoring [32P]-Rub1 transfer from Ubc12 to Cdc53C+ with the indicated versions of Hrt1 and Ubc12.
Figure 2
Figure 2. Synergistic functions of Dcn1P and Hrt1 as Rub1 E3s
Error bars - +/−1 standard deviation from 3 independent experiments. A. Phosphorimager data (left) and quantification (right) for time-course of pulse-chase assay monitoring [32P]-Rub1 transfer from Ubc12 to Cdc53C+ in the presence or absence of Dcn1P and the indicated versions of Hrt1 either in complex with Cdc53C+ or added in trans. To observe comparable levels of Rub1 ligation, a shorter time-course is shown for wild-type Cdc53C+-Hrt1 in the presence of Dcn1P. B. Time course of Ubc12~Rub1 discharge to hydroxylamine as in Fig. 1E, except in the presence or absence of Dcn1P, for full-length Ubc12 or the isolated core domain (Ubc12core). Raw data – left; quantification – right. C. Same assay as in (A), but comparing the activity of full-length Ubc12 or the Ubc12 core domain. Raw data – left; quantification – right. Note different times. D. Same assay as in Fig. 1H, except with earlier time-points and in the presence of Dcn1P.
Figure 3
Figure 3. A high affinity interaction between Dcn1P and the Cdc53 WHB subdomain
A. Schematic views of Cdc53C+, Dcn1, and Hrt1 domains. B. Summary of thermodynamic parameters determined by ITC for binding between the indicated Cdc53-Hrt1 and Dcn1 variants. C. Overall crystal structure of Dcn1P (violet)-Cdc53WHB (lime). The location of the Rub1 acceptor Lys760 is indicated in sticks, with a sphere for the ε-amino group.
Figure 4
Figure 4. E3 interactions for Rub1 ligation to Cdc53
A. Close-up view crystal structure of Dcn1P (violet)-Cdc53WHB (lime), with contact residues shown in sticks and electrostatic interactions as dashed lines. B. ITC data for the D226A/D259A double mutant Dcn1P and Cdc53C+-Hrt1. C. Phosphorimager data from multiple turnover experiments examining time-courses for forming [32P]-Rub1~Cdc53-Hrt1 in the presence of Ula1-Uba3 (Rub1 E1), Ubc12, and the indicated versions of Dcn1P. D. Cartoon view of structural model of dual Rub1 E3 ligase (Dcn1P, violet / Hrt1, blue with zinc atoms in grey) with Ubc12 (cyan) and Cdc53C+ (lime), generated as shown in Fig. S4. The 4 N-terminal residues of Ubc12 and residues surrounding the Dcn1 groove, which are mutated in Fig. 5, are shown in red. For simplification, Rub1, which would start thioester bound to Ubc12 C115 and end ligated via an isopeptide bond to Cdc53 Lys760 is not shown.
Figure 5
Figure 5. Mutational analysis of Ubc12 N-terminal helix and Dcn1 groove in Rub1 ligation to Cdc53
A. Phosphorimager data (left) and quantification (right) for time-course of pulse-chase assay monitoring [32P]-Rub1 transfer to Cdc53C+ from the indicated versions of Ubc12 in the presence or absence of Dcn1P. To observe comparable levels of Rub1 ligation, shorter time-courses are shown in the presence of Dcn1P. Error bars - +/− 1 standard deviation from 3 independent experiments. B. Same as (A), but with Ala or Pro mutations in place of Ubc12 residues 8 and 9. Note different time courses. C. Phosphorimager data for time-course of pulse-chase assay monitoring [32P]-ubiquitin (Ub) transfer from UbcH5b or a chimeric version (Ubc12N/UbcH5b, with Ubc12 residues 1–24 grafted at the N-terminus of UbcH5b) to Cdc53C+ in the absence or presence of Dcn1P. D. Phosphorimager data for time-course of pulse-chase assay monitoring [32P]-Rub1 transfer from Ubc12 to Cdc53C+ in the presence of the indicated versions of Dcn1P. E. Phosphorimager data (left) and quantification (right) showing effects of a peptide (Ubc12N), corresponding to Ubc12 residues 1–24 on inhibition of [32P]-Rub1 transfer to Cdc53C+ from full-length or the core domain versions of Ubc12, in the presence or absence of Dcn1P. To observe comparable levels of Rub1 ligation, shorter time-courses are shown in the presence of Dcn1P. Error bars - +/− 1 standard deviation from 2 independent experiments. F. Assay as in (E), but with wild-type Ubc12 and the indicated versions of Dcn1P. Note that longer time-courses were used for Dcn1P groove mutants to better depict their lack of inhibition by Ubc12N. In quantification (right): *Orange line – effects of adding the Ubc12N peptide in the presence of the E105A/D106A double mutant of Dcn1P. Remainder: data for experiments with the D89A/D91A and D185A/E186A double mutants of Dcn1P, either in the presence or absence of Ubc12N peptide. Error bars - +/− 1 standard deviation from 2 independent experiments.
Figure 6
Figure 6. Functions of the Dcn1 Rub1 E3
Error bars - +/− 1 standard deviation from 3 independent experiments. A. Michaelis-Menten curves for pulse-chase [32P]-Rub1 transfer to Cdc53C+-Hrt1, from Ubc12 Asn107Ala, in the absence or presence of Dcn1P. B. Kinetic constants obtained from data in (A). C. Bis-Maleimidoethane (BMOE) crosslinking (X) of Ubc12C115only (full-length or core domain) to Cdc53C+/K760C, in the absence or presence of Dcn1P or the D226A/D259A double mutant (Dcn1PM). Shown are western blots with antibodies against Cdc53 (top) or Ubc12 (bottom).
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