1. Volume 34, Issue 6, Pages 674-685 (June 2009)
Allosteric Activation of E2-RING Finger-Mediated Ubiquitylation by a Structurally Defined Specific E2-Binding Region of gp78 
Ranabir Das, Jennifer Mariano, Yien Che Tsai, Ravi C. Kalathur, Zlatka Kostova, Jess Li, Sergey G. Tarasov, Robert L. McFeeters, Amanda S. Altieri, Xinhua Ji, R. Andrew Byrd, Allan M. Weissman 
Molecular Cell 
Volume 34, Issue 6, Pages (June 2009)
DOI: /j.molcel
Copyright © 2009 Elsevier Inc. Terms and Conditions

2. Figure 1 The gp78 G2BR and Its Interactions with Ube2g2 by NMR
(A) Schematic representation of gp78 in the ER membrane (left). To the right is a linear representation of gp78 cytoplasmic tail with amino acids (corresponding to the entire human gp78) indicated. Peptides used in this study corresponding to the G2BR, specific mutations/truncations, and a scrambled (Scr) peptide control are shown below. Mutations are indicated in lowercase.
(B) Overlay of 15N-HSQC NMR spectra of Ube2g2 in free (red) and G2BR-bound (blue) form.
(C) The contact residues observed in an intermolecular NOESY spectrum of Ube2g2:G2BR are painted blue on the free Ube2g2 structure (PDB entry 2CYX). Region of E1 and E3 binding based on other E2-E3 pairs is indicated by the bracket. The active site Cys (C89) is in red.
(D) 15N-HSQC of isotopically labeled G2BR in free form (red) and bound to Ube2g2 (blue).
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Crystal Structure of the Ube2g2:G2BR Complex
(A) Ribbon representation of the superimposed Ube2g2:G2BR complex with free Ube2g2 (PDB entry 2CYX). G2BR is cyan, and Ube2g2 is light green in the complex. Free Ube2g2 is shown in orange.
(B) Linear representation of the secondary structure and binding regions of Ube2g2. α helices are represented by open rectangles; β strands, by filled arrows; and the active site Cys, by a red dot. Binding regions are indicated below the sequence line as RING finger (magenta) and G2BR (green). The position of Ube2g2's extended dynamic loop is indicated in orange.
(C) Hydrophobic side chains of G2BR (light blue, residues in black) lock into the hydrophobic surface of Ube2g2 (dark green, residues in yellow).
(D) Intermolecular hydrogen bonds and salt bridges between G2BR and Ube2g2 are shown. Side chains of G2BR are shown in blue; residues, in black; the Ube2g2 contact side chains and residues, in red. The P21 and I24 side chains of Ubeg2g2 were not displayed because the interaction is through backbone hydrogen bonds.
(E) Contacts between Ube2g2 (green) and G2BR (blue) indicating residues involved in hydrogen bonds, salt bridges, and hydrophobic interactions. Black lines link each residue to its reciprocal contact, and X denotes G2BR residues that do not have direct contacts in the interface.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
G2BR-Induced Structural Changes in Ube2g2 Occlude the Area around the Active Site and Correlate with Ubiquitin Loading
(A) Superposition of active site region (full structures shown in small inset) for four Ube2g2 molecules (yellow, cyan, and magenta from the ligand-free Ube2g2 in PDB entry 2CYX and green in Ube2g2:G2BR) showing the conformational flexibility of the β4α2 loop.
(B and C) Images showing the surface rendering of Ube2g2 and Ube2g2:G2BR combined with the potential orientation of ubiquitin chains, based on known E2-Ub structures. Ube2g2 was superimposed onto the E2 coordinates of two published E2-Ub structures: Ubc1-Ub (PDB entry 1FTX, rmsd = 1.8Å) and Ubc13-MMs2-Ub (PDB entry 2GMI, rmsd = 1.4Å). We display only the surface of Ube2g2 and the C-terminal end of the ubiquitin molecule (in ball and stick form) for Ubc1-Ub (blue) and Ubc13-MMs2-Ub (magenta). In (B), the free Ube2g2 structure is rotated relative to the orientation of Figure 1C by 180° about the vertical axis and zoomed in on the active site. The active site Cys (C89) backbone is yellow, and its side chain is red. Some residues of Ube2g2 that play a role in the allosteric change are labeled in green. R74 of both ubiquitin tails are indicated; the C-terminal diglycine approaches C89. In (C), the G2BR-bound form of Ube2g2 is shown in an identical orientation as in (B).
(D) 35S-labeled Ube2g2 or Ube2g2Δ96–108 generated by in vitro translation in E. coli lysate was incubated with 100 nM E1 and ubiquitin-lacking lysines (Ub K0), so as to avoid formation of polyubiquitin chains. The formation of thiolester-linked Ube2g2 (E2∼Ub) was assessed at 30°C with saturating (4 μM) G2BR peptide or a control “scrambled” peptide (Scr). Shown is the average of two experiments for each condition. Rate constant, K, and 95% confidence index are shown for each condition.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Interactions between gp78 RING Finger and Ube2g2:G2BR Complex
(A) Ube2g2:G2BR complex was titrated with gp78 RING finger followed by acquisition of 15N-HSQC spectra of isotopically labeled Ubeg2g2 at each titration point. 15N chemical shifts in parts per million (ppm) are indicated on the y axis, and 1H chemical shifts in ppm are on the x axis. Three of the affected residues of Ube2g2 (N19, L66, and V113) are shown. The peaks shift from the free (blue) form toward the bound form (magenta) with addition of gp78 RING finger. The dissociation constant was determined by fitting the peak positions against ligand: protein concentrations.
(B) The surface representation of Ube2g2:G2BR shows the G2BR interface (blue), RING interface (magenta), and active site cysteine (red).
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
G2BR-Mediated Increase in Ube2g2:gp78 RING Finger Affinity Results in Enhanced Ubiquitylation
(A) Glutathione Sepharose-bound GST fusions of the entire gp78 cytoplasmic tail (aa 309–643; GST-gp78C), an inactivating mutation in the RING finger (GST-gp78Crm) or in the G2BR (GST-gp78Cl582,589s), or GST alone were incubated for 90 min in the presence of Ube2g2 and E1. After washing, ubiquitylated bead-bound material was assessed by SDS-PAGE and immunoblotting with antiubiquitin. See Figure S10 for Coomassie blue stain of fusion proteins.
(B) Glutathione Sepharose-bound GST-gp78C, a truncation at amino acid 577 at the beginning of the G2BR (GST-gp78CΔ577–643), or GST alone was incubated with or without G2BR peptide as indicated and assessed in (A).
(C and D) (C) and (D) were carried out as in (A) using GST-gp78CΔ577–643 and the indicated synthetic or recombinant peptides. Recombinant wild-type G2BR peptide is indicated by an asterisk to distinguish from the synthetic wild-type peptide.
(E) Ubiquitylation of Ub-GFP-His6 was carried out under the indicated conditions. The asterisk denotes two control samples in which Ub-GFP-His6 was added after the reaction was first terminated by the addition of 10% SDS for 10 min followed by 8 M urea. Following addition of urea, Ub-GFP-His6 was purified on Ni+ Sepharose beads, and samples were resolved by SDS-PAGE.
(F) Ubiquitylation of Ub-GFP-His6 was carried out as in (E) for 60 min.
(G) Ubiquitylation reactions were carried out with GST-gp78C or the indicated Leu to Ser mutations together with wild-type Ube2g2 or the indicated double mutations of Ube2g2.
(H) GST fusions of the cytoplasmic tails of HsHRD1, Trc8, or truncated gp78 were incubated as in (A) with the indicated peptides and E2s.
(I) 35S-labeled Ube2g2 generated as in Figure 3D was loaded with wild-type ubiquitin for 10 min followed by inactivation of E1 by NEM (5 mM). Discharge of ubiquitin from Ube2g2 (<100 nM), in the presence of either G2BR or Scr (4 μM) with or without gp78 RING finger (8 μM), was monitored by measuring the fraction of Ube2g2∼Ub remaining at each time point. Shown on the left is a representative experiment using gp78 RING finger. Plotted on the right is the average of three independent experiments; error bars represent SD.
(J) Discharge experiments as in (I) were carried out for 2 or 5 min at the indicated concentrations of gp78 RING finger. Shown on the left are images demonstrating loss of ubiquitin from Ube2g2. The insert on the right summarizes data from discharge experiments from both (I) and (J). Discharge rate constants, K, are shown for each experiment.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions