Kuriyan Lab Logo      Katherine Wickliffe      Sonja Lorenz      Stamp


The Mechanism of Linkage-Specific Ubiquitin Chain Elongation by a Single-Subunit E2


Katherine E Wickliffe, Sonja Lorenz, David E. Wemmer, John Kuriyan, and Michael Rape
∗ These authors contributed equally to this work


Cell Volume 144, Issue 5, 4 March 2011, Pages 769-781 doi:10.1016/j.cell.2011.01.035 (local copy)

Abstract / Figures from the paper / Tables from the paper / References



Abstract:

Abstract Figure Ubiquitin chains of different topologies trigger distinct functional consequences, including protein degradation and reorganization of complexes. The assembly of most ubiquitin chains is promoted by E2s, yet how these enzymes achieve linkage specificity is poorly understood. We have discovered that the K11-specific Ube2S orients the donor ubiquitin through an essential noncovalent interaction that occurs in addition to the thioester bond at the E2 active site. The E2-donor ubiquitin complex transiently recognizes the acceptor ubiquitin, primarily through electrostatic interactions. The recognition of the acceptor ubiquitin surface around Lys11, but not around other lysines, generates a catalytically competent active site, which is composed of residues of both Ube2S and ubiquitin. Our studies suggest that monomeric E2s promote linkage-specific ubiquitin chain formation through substrate-assisted catalysis.


Figures from the paper.



Click on the small image to get a bigger one.


First figure from paper

Figure 1: Ube2S Recognizes the Hydrophobic Patch of Donor Ubiquitin

(A) Overview of K11-specific linkage formation. Lys11 of acceptor ubiquitin attacks the thioester bond between Cys95 of Ube2S and the C terminus of the donor ubiquitin.

(B) Ube2S interacts with ubiquitin noncovalently. Weighted combined chemical shift perturbations, Δδ(1H15N), are plotted over residue number. The asterisk indicates the disappearance of the resonance for His68 of ubiquitin in the presence of Ube2S due to intermediate exchange.

(C) Mutation of the hydrophobic patch in ubiquitin interferes with formation of K11-linked ubiquitin dimers (ubi~ubi) by Ube2S, as monitored by Coomassie staining.

(D) Ube2S and APC/C extend ubiquitin chains on a fusion between ubiquitin and cyclin A (Ub-L-cycA), as analyzed by autoradiography.

(E) The hydrophobic patch of ubiquitin is required for chain elongation by APC/CCdh1 and Ube2S, as analyzed by autoradiography.

(F) The hydrophobic patch is not required on acceptor ubiquitin. Ube2S was mixed with acceptor His6ubiquitinΔGG mutants (ubiΔGG; purple) and WT-ubiquitin (blue) and analyzed by α-ubiquitin-western.

(G) The hydrophobic patch is required on the donor ubiquitin. Ube2S was mixed with WT-ubiΔGG and ubiquitin mutants and analyzed by α-ubiquitin-western.

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First supplimental figure from paper

Figure S1: Characterization of Ube2S and Its Interaction with Donor Ubiquitin, Related to Figure 1

(A) The UBC domain of Ube2S (UBCUbe2S) promotes formation of K11 linkages between ubiquitin molecules. UBCUbe2S or Ube2S were incubated with E1, ubiquitin or ubiK11R, and ATP for the indicated times. Reaction products were analyzed by SDS-PAGE and Coomassie staining.

(B) Radius of gyration, Rg, of Ube2S and UBCUbe2S at various protein concentrations, as derived from Guinier analysis in PRIMUS (Konarev et al., 2003). Analysis with the indirect transform package GNOM (Svergun et al., 1988) yielded similar Rg values (data not shown). For both proteins, Rg does not change significantly with increasing protein concentration, consistent with a lack of oligomerization. The Rg-values of ~17 and 28 Å correlate well with the hydrodynamic radius/molecular mass of the monomeric states of UBCUbe2S and Ube2S, respectively, as derived by gel filtration and multi-angle light scattering (data not shown).

(C) Comparison of experimental and simulated scattering curves generated with CRYSOL (Svergun, D., Barberato, C., and Koch, M.J.H. (1995). CRYSOL- A program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates. J. Appl. Cryst. 28, 768-773.Svergun et al., 1995) based on a UBCUbe2S monomer (PDB ID: 1ZDN, chain A) and a crystallographic dimer (PDB ID: 1ZDN, both chains). I(q) is plotted as a function of the momentum transfer q = (4π*sin(Θ))/λ), where 2Θ is the scattering angle and λ is the wavelength of the incident x-ray beam.

(D) The hydrophobic patch of ubiquitin is required for Ube2S- and APC/C-dependent chain formation over a wide range of ubiquitin concentrations. Ub-L-cycA was produced by IVT/T and incubated with APC/CCdh1, Ube2S, E1, ATP, and the indicated concentrations of either WT-ubiquitin or ubiI44A. The formation of K11-linked ubiquitin chains on Ub-L-cycA was monitored by SDS-PAGE and autoradiography. As a comparison, the concentration of ubiquitin in HeLa cells has been estimated at 90 μM (Ryu et al., 2006).

(E) The hydrophobic patch in ubiquitin is required for APC/C-dependent ubiquitin chain elongation. 35S-labeled APC/C substrate cyclin A was incubated with APC/CCdh1, E1, low concentrations of Ube2C (for chain initiation) and Ube2S (for chain elongation). Reaction products were separated by SDS-PAGE and analyzed by autoradiography. Due to the presence of Ube2S, the majority of modified cyclin A is decorated with long ubiquitin chains.

(F) The hydrophobic patch of ubiquitin is not required for charging of Ube2S by the E1. Ube2S was incubated with E1 and ubiquitin or the indicated ubiquitin mutants in the absence of reducing agents. When indicated, β-mercaptoethanol was added to gel-loading buffer to reduce thioester linkages. Charging of Ube2S with ubiquitin results in a βME-sensitive conjugate representing the thioester (Ube2S-Cys95~ubi) and in a βME-insensitive conjugate, most likely a covalent modification of a lysine residue in the UBC domain of Ube2S (Ube2S-ubi).

(G) A functional hydrophobic patch is not required in the acceptor ubiquitin. The L8A and I44A/V70A-mutations were introduced into the acceptor ubiΔGG. ubiΔGG and indicated mutants were mixed with ubiquitin, E1, ATP, and Ube2S, and the formation of ubiΔGG-ubi and ubi-ubi dimers was monitored by SDS-PAGE and Silver staining.

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Second figure from paper

Figure 2: Noncovalent Donor Ubiquitin Binding Is Required for Ube2S Activity

(A) Identification of Ube2S residues involved in noncovalent binding of ubiquitin. Weighted combined chemical shift perturbations, Δδ(1H15N), are plotted over the residue number.

(B) Donor binding is required for formation of ubi2 dimers (ubi~ubi) by Ube2S mutants, as analyzed by Coomassie staining.

(C) Donor binding by Ube2S is required for chain elongation on Ub-L-cycA with APC/C, as analyzed by autoradiography.

(D) Donor binding by Ube2S is required for chain formation in a full APC/C assay. Ubiquitination of cyclin A by APC/C, Ube2C, and Ube2S mutants was analyzed by autoradiography.

(E) Donor binding is required for Ube2S activity in vivo. HeLa cell lines expressing Ube2S or Ube2SI121A were treated with siRNAs against the 3' UTR of Ube2S, which specifically depletes endogenous Ube2S. Cells were synchronized in prometaphase (t = 0 hr) or late mitosis (t = 2 hr), and K11-linked ubiquitin chains were detected by αK11-western.

(F) Donor binding occurs in cis. Ube2SC118A or Ube2SI121A (lack the noncovalent ubiquitin-binding site) and Ube2SC95A (no active site) were mixed, and ubi2 formation was monitored by Coomassie staining.

See also Figure S2.

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Second supplimental figure from paper

Figure S2: Characterization of Donor Ubiquitin Binding to Ube2S, Related to Figure 2

(A) Ubiquitin causes similar chemical shift perturbations on UBCUbe2S regardless of whether it is added in trans or is covalently linked to the active site. To obtain a more stable oxy-ester complex, the C95S mutant of UBCUbe2S was used instead of WT. Weighted combined chemical shift perturbations, Δδ(1H15N), are plotted over the residue number. 1H-15N HSQC spectra were recorded of 22 μM 15N-enriched UBCUbe2S C95S ester-linked to unlabeled ubiquitin (yellow) and 140 μM 15N-enriched UBCUbe2S C95S in the presence of an 11-fold molar excess of unlabeled ubiquitin (red) and were referenced to the spectrum of 140 μM 15N-enriched UBCUbe2S C95S in the absence of ubiquitin. Gaps are due to proline residues or missing assignments.

(B) Residues with significant binding-induced chemical shift perturbations and significant surface accessibility (see Table S1) are mapped onto the surface of UBCUbe2S (PDB ID: 1ZDN) and ubiquitin (PDB ID: 1UBQ), respectively.

(C) Mutations in the UBCUbe2S-donor ubiquitin interface interfere with the interaction detected by NMR. Weighted combined chemical shift perturbations, Δδ(1H15N), are plotted over residue number. The data are based on 1H-15N HSQC spectra of mixtures of 200 μM 15N-enriched ubiquitin and a 6-fold molar excess of UBCUbe2S. Gaps are due to proline residues or missing assignments. As justified under Materials and Methods, we interpret the amplitude of Δδ(1H15N) as a measure of binding affinity.

(D) Determination of the dissociation constants, KD, for the interaction between Ube2S and ubiquitin in solution. NMR-derived isotherms for the binding of ubiquitin to Ube2S (left panel) and UBCUbe2S (right panel) were fitted globally to a single-site model. Only those resonances were included that show a weighted combined chemical shift perturbation, Δδ(1H15N), of at least 0.05 ppm at the highest excess of ubiquitin used. The concentration of Ube2S and UBCUbe2S was 240 μM.

(E) Mutations in the noncovalent donor ubiquitin binding interface of Ube2S do not inhibit charging of Ube2S by E1. Ube2S or indicated mutants were incubated with ubiquitin E1 and ATP, resulting in formation of a βME-sensitive thioester (Ube2S-Cys95~ubi) and a βME-insensitive conjugate (Ube2S-ubi). Reactions were analyzed by αUbe2S-western.

(F) Mutations of Ube2S do not interfere with binding of Ube2S to the APC/C. APC/C subunits were synthesized by in vitro-transcription/translation. The radiolabeled proteins are incorporated into full APC/C present in reticulocyte lysate (our unpublished observations). The 35S-labeled proteins were then incubated with MBPUbe2S, which was immobilized on amylose-resin; MBP was used as a control. After extensive washing, binding reactions were analyzed by SDS-PAGE and Coomassie staining (for inputs; bottom panel) or autoradiography (for binding; top panel).

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Third figure from paper

Figure 3: Structural Model of the Ube2S-Donor Ubiquitin Complex

(A) NMR-based HADDOCK model of the UBCUbe2S-donor ubiquitin complex (cluster 1, no. 3; see Table S1). The C-terminal tail of ubiquitin (cyan) was allowed full flexibility during docking.

(B and C) Surface representation of the binding interface on UBCUbe2S (B) and donor ubiquitin (C). Residues that make intermolecular contacts within a radius of 4 Å are shown in pink.

(D) Ube2S residues at the donor-binding interface are required for formation of ubi2 dimers (ubi~ubi), as monitored by Coomassie staining.

(E) Donor-binding-deficient Ube2S mutants do not promote chain elongation on Ub-L-cycA with APC/C, as analyzed by autoradiography.

(F) Ubiquitin residues at the Ube2S interface are required for linkage formation, as seen by Coomassie staining.

(G) Ubiquitin residues at the Ube2S interface are required for chain elongation on Ub-L-cycA with APC/C, as analyzed by autoradiography.

(H) Donor binding is required for Ube2S activity in cells. HeLa cell lines expressing donor-binding-deficient Ube2S (E51K; D102A; S127A) were depleted of endogenous Ube2S, synchronized in prometaphase (t = 0) or allowed to exit mitosis (t = 2 hr), and tested for K11-linked ubiquitin chains by αK11-western.

(I) Charge-swap analysis of the ionic interaction between Lys6 of donor ubiquitin and Glu51 on Ube2S. ubiΔGG was mixed with ubiquitin or ubiK6E in the presence of Ube2S or Ube2SE51K. Reactions were monitored by Silver staining.

See also Figure S3 and Table S1.

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Third supplimental figure from paper

Figure S3: Characterization of the NMR-Based Model of the Ube2S-Donor Ubiquitin Interface, Related to Figure 3

(A) Comparison of docked models generated by two different programs, HADDOCK, including NMR-based restraints (blue/yellow), and ClusPro without any restraints (green). Major differences are only seen for the C-terminal tail of ubiquitin, which is highlighted red in the ClusPro model. The chosen ClusPro model represents 9 out of 965 models generated, 38 of which have the Sγ atom of Cys95 of Ube2S within a distance of 7.5 Å from the C-terminal carbon atom of donor ubqiutin.

(B) Surface electrostatic potentials of the donor interface, as calculated using APBS ([Baker et al., 2001], [Dolinsky et al., 2004] and [Dolinsky et al., 2007]). Intermolecular salt bridges, as predicted by the PISA server at the European Bioinformatics Institute (http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html; Krissinel et al., 2007), are illustrated by solid lines.

(C) Illustration of the ionic contact between Arg74 of donor ubiquitin and Asp102 on Ube2S, as seen in our model of the Ube2S-donor ubiquitin complex.

(D) Residues in ubiquitin that are at the Ube2S-donor ubiquitin interface are not required on the acceptor ubiquitin. Mutations were introduced into the acceptor ubiΔGG. ubiΔGG and indicated mutants were incubated with E1, Ube2S, ubiquitin, and ATP, and formation of ubiΔGG-ubi and ubi-ubi dimers was monitored by SDS-PAGE and Silver staining. Except for Lys6, no residue was required in the acceptor ubiquitin.

(E) Lys6 is required in the donor ubiquitin. Ubiquitin and the respective mutants K6A and K6E were incubated with the acceptor ubiΔGG, E1, Ube2S, and ATP. Reaction products were analyzed by SDS-PAGE and Coomassie staining. Mutation of Lys6 to Glu affects both acceptor and donor ubiquitin, and thus, neither ubiΔGG-ubi nor ubi-ubi dimers are formed. Mutation of Lys6 to Ala only affects acceptor ubiquitin function, and thus, ubiuΔGG-ubi dimers are formed with this mutant. Both ubiK6E and ubiK6A showed higher mobility in SDS-PAGE compared to WT-ubiquitin.

(F) With exception of Arg72, all ubiquitin residues at the Ube2S-donor ubiquitin interface can be mutated without affecting Ube2S-charging by E1. Ube2S was incubated with ubiquitin or indicated mutants, E1, and ATP, resulting in formation of a βME-sensitive thioester (Ube2S-Cys95~ubi) and a βME-insensitive conjugate (Ube2S-ubi). Reaction products were analyzed by SDS-PAGE and αUbe2S-western.

(G) Ube2S-residues at the binding interface with donor ubiquitin are not required for charging of Ube2S by E1. Ube2S and indicated mutants were incubated with E1, ubiquitin, and ATP. Reaction products were analyzed by SDS-PAGE and αUbe2S-western.

(H) The E51K mutant of Ube2S does not rescue defective ubiquitin dimer formation in the presence of the I44A-mutation on ubiquitin. Ubiquitin or ubiiI44A were incubated with acceptor ubiΔGG, Ube2S or Ube2SE51K, E1, and ATP. Reaction products were analyzed by SDS-PAGE and Silver staining.

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Fourth figure from paper

Figure 4: Noncovalent Donor Binding Increases the Processivity of Ube2S

(A) Donor binding is not required for the K11-linkage specificity of Ube2S. Ube2S or donor-binding mutants were incubated with ubiquitin or ubiK11R, or as indicated with ubiI44A and ubiI44A/K11R. Reactions were incubated longer and at higher ubiquitin concentrations to observe formation of ubi2 and analyzed by Coomassie staining.

(B) Donor binding promotes catalysis at low acceptor concentrations. Dimer formation between increasing levels of ubiΔGG and ubiK11R or ubiK11R/I44A, respectively, by Ube2S was monitored by Silver staining.

(C) Time-course analysis of chain elongation on Ub-L-cycA by APC/CCdh1 and Ube2S in the presence of ubiquitin mutants, as analyzed by autoradiography.

(D) Donor binding is required for processive chain formation by Ube2S. Chain elongation on Ub-L-cycA by APC/C and Ube2S was monitored in the presence of the UBA domains of Rad23A. Reactions were performed with ubiquitin or ubiV70A and analyzed by autoradiography (top) and line scanning (bottom).

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Fourth supplimental figure from paper

Figure S4: Characterization of Donor Ubiquitin Recognition by Ube2R1 and Ube2G2, Related to Figure 5

(A) The hydrophobic patch of ubiquitin is required for ubiquitin-dimer formation by the K48-specific E2s Ube2G2 (top panel) and Ube2R1 (bottom panel). Ubiquitin and indicated mutants were incubated with Ube2R1 or Ube2G2/gp78, E1, and ATP. Reaction products were separated by SDS-PAGE and analyzed by Coomassie staining.

(B) The hydrophobic patch of ubiquitin is not required for charging of Ube2G2 (top panel) or Ube2R1 (bottom panel) by E1. Ubiquitin and indicated mutants were incubated with E1, ATP, and Ube2G2 or Ube2R1 in the absence of reducing agents. Where indicated, βME was added to gel loading buffer to show thioester formation. Reaction products were monitored by αHis-western, detecting a His-epitope used to purify the E2 proteins.

(C) The same surface used by Ube2S for donor ubiquitin-binding is required on Ube2R1 for E2 activity. Ube2R1 or indicated mutants were incubated with E1, ubiquitin, and ATP. Formation of K48-linked ubiquitin dimers (ubi~ubi) was monitored by SDS-PAGE and Coomassie staining.

(D) Ube2R1 residues at the donor ubiquitin binding interface are not required for charging of Ube2R1 by the E1. Ube2R1 or indicated mutants were incubated with E1, ATP, and ubiquitin, and analyzed for charging as described above.

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Fifth figure from paper

Figure 5: Noncovalent Donor Binding Is Utilized by E2s Independently of Linkage Specificity

(A) Ube2R1 and Ube2G2 require the hydrophobic patch in the donor but not acceptor ubiquitin for K48-linkage formation. Ube2S, Ube2R1, or Ube2G2 and its E3 gp78 were incubated with ubiΔGG or ubiΔGG/I44A/V70A (purple) and ubiquitin or ubiI44A (blue). Reactions were analyzed by Silver staining.

(B) The hydrophobic patch of donor ubiquitin is not required for K48 specificity of Ube2R1 or Ube2G2. ubi2 formation by Ube2R1 or Ube2G2/gp78 with ubiquitin mutants was analyzed by Coomassie staining.

(C) Donor binding is required for rapid catalysis by Ube2R1. Time courses of ubi2 formation by Ube2R1 in the presence of increasing concentrations of ubiquitin or ubiI44A were analyzed by Coomassie staining.

(D) A similar surface as the donor-binding interface of Ube2S (yellow) is required in Ube2R1 (green; PDB ID: 2OB4). Ube2R1 mutants were analyzed for K48-specific ubi2 formation by Coomassie staining.

(E) Donor binding is required for processive chain formation by SCFβTrCP and Ube2R1. Phosphorylated IκBα was incubated with SCFβTrCP, Ube2R1 or Ube2R1I128E, and ubiquitin or ubiI44A/V70A and analyzed by autoradiography.

See also Figure S4.

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Fifth supplimental figure from paper

Figure S5: HADDOCK Output for the Docking of Acceptor Ubiquitin onto the UBCUbe2S-Donor Ubiquitin Complex, Related to Figure 6

Cartoon representation of the top-scoring models of the two clusters of run 1 without experimental restraints (see Table S2).

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Sixth figure from paper

Figure 6: Acceptor Ubiquitin Recognition by the Ube2S-Donor Ubiquitin Complex

(A) The TEK-box in ubiquitin is required for K11-linkage formation by Ube2S. Ube2S was incubated with ubiquitin mutants, and ubi2 formation (ubi~ubi) was monitored by Coomassie staining.

(B) TEK-box mutants in ubiquitin inhibit chain formation on Ub-L-cycA by Ube2S and APC/C, as analyzed by autoradiography.

(C) The TEK-box is required on acceptor ubiquitin. ubiΔGG mutants (purple) were incubated with ubiquitin (blue) and Ube2S and analyzed by Silver staining.

(D) The TEK-box is not required in donor ubiquitin. TEK-box mutants of ubiquitin were mixed with WT-ubiΔGG and Ube2S, and reactions were analyzed by Coomassie staining.

(E) HADDOCK-based model of the ternary complex between the UBCUbe2S (yellow), donor ubiquitin (blue), and acceptor ubiquitin (pink; cluster 1, no. 1; see Table S2, bottom).

(F) Surface representation of the Ube2S-binding interface on acceptor ubiquitin. Contact residues within a radius of 4 Å are shown in pink.

(G) Surface representation of the acceptor-binding interface on the Ube2S-donor complex.

(H) Acceptor binding is required for Ube2S activity. Ube2S mutants were incubated with ubiquitin and analyzed by Coomassie staining.

(I) Ube2S residues at the acceptor interface are required for chain elongation by APC/C. The modification of Ub-L-cycA by APC/C and Ube2S mutants was analyzed by autoradiography.

(J) Ube2S residues at the acceptor-binding interface are required in vivo. HeLa cell lines expressing Ube2SE131K and Ube2SR135E were treated with siRNAs to deplete endogenous Ube2S, arrested in prometaphase (t = 0 hr) or allowed to exit mitosis (t = 2 hr), and tested for K11-linked chains by αK11-western.

See also Figure S5, Figure S6, and Table S2.

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Sixth supplimental figure from paper

Figure S6: Characterization of Acceptor Ubiquitin Recognition by the Ube2S-Donor Ubiquitin Complex, Related to Figure 6

(A) The TEK-box of ubiquitin is required for APC/C-dependent chain formation catalyzed by the E2s Ube2C and Ube2S. Ubiquitin or indicated mutants were incubated with 35S-labeled cyclin A, APC/CCdh1, Ube2C, Ube2S, E1, and ATP. Reaction products were separated by SDS-PAGE and analyzed by autoradiography.

(B) The TEK-box residues of ubiquitin are not required for charging of Ube2S by E1. Ubiquitin or indicated mutants were incubated with E1, Ube2S, and ATP, and analyzed for charging as described above.

(C) Mutations in the Ube2S-acceptor ubiquitin interface do not influence the interaction between Ube2S and donor ubiquitin, as detected by NMR. Weighted combined chemical shift perturbations, Δδ(1H15N), are plotted over residue number. The data are based 1H-15N HSQC spectra of mixtures of 200 μM 15N-enriched ubiquitin and a 6-fold molar excess of UBCUbe2S. Gaps are due to proline residues or missing assignments.

(D) Surface electrostatic potentials of the acceptor interface for the selected HADDOCK complex between Ube2S, donor, and acceptor ubiquitin (cluster 1, no. 1; Table S2, bottom), as calculated using APBS ([Baker et al., 2001], [Dolinsky et al., 2004] and [Dolinsky et al., 2007]). Intermolecular salt bridges and hydrogen bonds, as predicted by the PISA server are illustrated by solid and dashed lines, respectively.

(E) Ube2S residues at the acceptor binding interface are required for APC/C-dependent ubiquitin chain formation. Ube2S or indicated mutants were incubated with 35S-labeled cyclin A, APC/CCdh1, E1, Ube2C, and ubiquitin, and reaction products were analyzed by SDS-PAGE and autoradiography.

(F) Ube2S residues at the acceptor binding interface are not required for charging of Ube2S by E1. Ube2S or indicated mutants were incubated with E1, ATP, and ubiquitin, and analyzed for charging as described above.

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Seventh figure from paper

Figure 7: Substrate-Assisted Catalysis Contributes to the K11-Linkage Specificity of Ube2S

(A) Charge-swap analysis of the ionic contact between acceptor Lys6 and Glu131 of Ube2S. Ube2S or Ube2SE131K was mixed with ubiΔGG or ubiΔGG/K6E and reactions were analyzed by Silver staining.

(B) Ube2SE51K/E131K rescues mutation of Lys6 in both acceptor and donor ubiquitin. Lys6 was mutated in acceptor ubiΔGG (purple) or donor ubiquitin (blue), and ubiΔGG-ubi formation by Ube2S or Ube2SE51K/E131K was analyzed by Silver staining.

(C) Ube2S (left) and Ubc9 (PDB ID: 2GRN; right) show similar active site constellations. The highest scoring Ube2S model of the HADDOCK run in the absence of ambiguous restraints is shown (Table S2, top; cluster 1, no 1).

(D) Candidate active-site residues are required for the activity of Ube2S to catalyze ubi2 formation (ubi~ubi), as analyzed by Coomassie staining.

(E) Active-site residues in Ube2S are required for chain elongation by APC/C. Ub-L-cycA was incubated with APC/CCdh1 and Ube2S mutants and analyzed by autoradiography.

(F) Leu129 is required for Ube2S activity in vivo. HeLa cell lines expressing Ube2S or Ube2SL129 were tested for formation of K11-linked chains after endogenous Ube2S was depleted by siRNAs. K11-chain formation in cells arrested in prometaphase or exiting mitosis was monitored by αK11-western.

(G) Glu34 of acceptor ubiquitin is required for K11-linkage formation. Ubiquitin mutants were incubated with Ube2S and analyzed by Coomassie staining.

(H) Glu34 of acceptor ubiquitin is required for chain elongation by APC/C and Ube2S. The modification of Ub-L-cycA by APC/C, Ube2S, and ubiquitin mutants was analyzed by autoradiography.

(I) ubiE34Q displays catalytic, but not binding defects. The rates of ubi2 formation at different concentrations of ubiquitin and ubiE34Q at the indicated pH were determined from two or three independent time courses. Apparent kinetic constants were obtained by fitting the rate constants to a Michaelis-Menten equation.

(J) Rescue of ubiE34Q, but not other TEK-box or hydrophobic patch mutants, by increasing the reaction pH. Ubiquitin or indicated mutants were incubated with Ube2S at pH 7.5 (left) or pH 9 (right) and analyzed by Coomassie staining.

See also Figure S7, Figure S8, and Table S3.

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Seventh supplimental figure from paper

Figure S7: Characterization of Acceptor Ubiquitin Recognition by Ube2S, Related to Figure 7

(A) The E131A mutant of Ube2S rescues the phenotype of the K6E mutation in acceptor ubiquitin. ubiΔGG or ubiΔGG/K6E were incubated with ubiquitin, E1, ATP, and Ube2S or indicated Ube2S mutants. Formation of ubiΔGG-ubi and ubi-ubi dimers was analyzed by SDS-PAGE and Coomassie staining. In contrast to Ube2SE131K, Ube2SE131A does not interfere with recognition of Lys6 in WT-ubiquitin, explaining the formation of ubi-ubi dimers in the presence of Ube2SE131A, but not Ube2SE131K.

(B) Specific rescue of the K6E-mutation in acceptor ubiquitin by Ube2SE131K, but not other mutants at the acceptor ubiquitin-binding interface. Ube2S or indicated mutants were incubated with E1, ATP, ubiΔGG or ubiΔGG/K6E, and ubiquitin. Formation of ubiΔGG-ubi and ubi-ubi dimers was analyzed by SDS-PAGE and Coomassie staining.

(C) Complete rescue of the ubiK6E-phenotype by Ube2SE51K/E131K. Ubiquitin or ubiK6E were incubated with Ube2S or Ube2SE51K/E131K, E1, and ATP, and formation of ubiquitin dimers was monitored by SDS-PAGE and Silver staining.

(D) Validation of the Ube2S-acceptor ubiquitin interaction by charge-swap analysis between Glu64 of ubiquitin and Arg135 of Ube2S. The indicated mutants of ubiquitin and Ube2S were tested for their ability to produce ubiquitin dimers (ubi~ubi). Reaction products were analyzed by SDS-PAGE and Coomassie staining.

(E) Specific rescue of the deleterious effects of a R135E mutant of Ube2S by a E64K mutant of ubiquitin. Ube2S or Ube2SR135E were incubated with ubiquitin or indicated TEK-box mutants, and formation of ubiquitin dimers (ubi~ubi) was monitored by SDS-PAGE and Coomassie staining.

(F) Rescue of impaired ubiquitin dimer formation by a K63E mutant of ubiquitin by Ube2SE139K. Ube2S or Ube2SE139K were incubated with ubiquitin or ubiK63E, E1, and ATP, and formation of ubiquitin dimers was analyzed by SDS-PAGE and Coomassie staining.

(G) Ube2S residues required for ubiquitin-linkage formation are not required for charging of Ube2S by the E1. Ube2S or indicated mutants were incubated with E1, ATP, and ubiquitin, and analyzed for charging as described above.

(H) Reduced substrate-specificity of Ube2S at higher pH. A peptide of 26 C-terminal residues of Ube2S tagged with biotin (BCTP) was incubated with Ube2S, E1, ATP, and ubiquitin at either pH 7.5 or at pH 9. Modification of lysine residues in BCTP was detected by western blotting using HRP-coupled streptavidin.

(I) Increasing the pH allows Ube2S to modify ubiquitin lysine residues other than K11. The indicated single lysine ubiquitin mutants were incubated with Ube2S, E1, and ATP at either pH 7.5 or pH 9. Formation of ubiquitin dimers (ubi~ubi) was detected by western blotting using an α-ubiquitin antibody.

(J) Increasing the pH does not completely obliterate the K11-specificity of Ube2S. Ubiquitin or the indicated mutants were incubated with Ube2S, E1, and ATP at pH 9. The formation of ubiquitin dimers (ubi~ubi) was monitored by SDS-PAGE and Silver staining.

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Eight supplimental from paper

Figure S8: HADDOCK Analysis of Ube2S-Donor-Acceptor Complexes Exposing Each of the Seven Lysine Residues of the Acceptor Ubiquitin to the Active Site of Ube2S, Related to Figure 7

Details of the active site are shown for the top-scoring model of each of seven HADDOCK runs in stereo representation with relevant side chains rendered as ball-and-stick. Only the C-terminal tail (residues 71-76) of donor ubiquitin is displayed (blue). Ribbons for acceptor ubiquitin and Ube2S are shown in pink and gray, respectively. While structures docked around K11 show a favorable active site constellation (see Figure 7C), complexes exposing other lysine residues have features incompatible with efficient catalysis. In the following we describe these features for the top representatives of the most populated clusters for each of seven HADDOCK runs; note, however, that these conclusions also hold for the lower-ranked clusters. Around K6 and K48 no acidic groups are found on the acceptor within a radius of ~8 Å and ~10 Å, respectively. K33 is neighbored by an acidic residue, E34; in this case, however, the acidic side chain points away from the active site, which puts K33 in an unfavorable orientation for the nucleophilic attack. Structures docked around K27 display productive active site geometry including a proximal acidic residue, D52. However, these complexes contain an additional acidic residue, D39, near the active site cysteine of Ube2S, which might interfere with catalysis by destabilizing the thiolate intermediate formed during the nucleophilic substitution reaction. Complexes docked around K29 contain D21 of the acceptor ubiquitin in a distance of ~5 Å from K29, but their overall geometry appears sterically unfavorable. K63 can be docked in a reasonable orientation, but the position of the neighboring acidic side chain, E64, appears less optimal than in the case of K11.
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Tables from paper


Tables from paper as pdf

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References


Please see paper.