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Allosteric Activation of E2-RING Finger-Mediated Ubiquitylation by a Structurally Defined Specific E2-Binding Region of Gp78

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Allosteric Activation of E2-RING Finger-Mediated Ubiquitylation by a Structurally Defined Specific E2-Binding Region of Gp78 Molecular Cell Article Allosteric Activation of E2-RING Finger-Mediated Ubiquitylation by a Structurally Defined Specific E2-Binding Region of gp78 Ranabir Das,1 Jennifer Mariano,2 Yien Che Tsai,2,4 Ravi C. Kalathur,3,4 Zlatka Kostova,2 Jess Li,1 Sergey G. Tarasov,1 Robert L. McFeeters,1 Amanda S. Altieri,1 Xinhua Ji,3 R. Andrew Byrd,1,* and Allan M. Weissman2,* 1Structural Biophysics Laboratory 2Laboratory of Protein Dynamics and Signaling 3Macromolecular Crystallography Laboratory Center for Cancer Research, National Cancer Institute, Frederick, MD 21702-1201, USA 4These authors contributed equally to this work *Correspondence: [email protected] (R.A.B.), [email protected] (A.M.W.) DOI 10.1016/j.molcel.2009.05.010 SUMMARY characterized by a conserved 350 amino acid catalytic domain. HECT E3s are catalytic intermediates in substrate ubiquitylation The activity of RING finger ubiquitin ligases (E3) is as a consequence of the transthiolation of ubiquitin from bound dependent on their ability to facilitate transfer of E2 to their conserved catalytic Cys (Fang and Weissman, 2004). ubiquitin from ubiquitin-conjugating enzymes (E2) RING finger and RING finger-like E3s collectively represent the to substrates. The G2BR domain within the E3 gp78 large majority of E3s. The RING finger is a compact Zn-binding binds selectively and with high affinity to the E2 domain of 40 to 100 amino acids. These domains generally Ube2g2. Through structural and functional analyses, bind E2s with low affinity and do not form catalytic intermediates with ubiquitin. It is generally believed that RING fingers either we determine that this occurs on a region of Ube2g2 position E2Ub to facilitate transfer to substrates or function distinct from binding sites for ubiquitin-activating as allosteric activators of E2Ub (Lorick et al., 2005; Ozkan enzyme (E1) and RING fingers. Binding to the G2BR et al., 2005). Binding sites on E2s for RING fingers, HECT results in conformational changes in Ube2g2 that domains, and E1 all overlap. Therefore, E2s must dissociate affect ubiquitin loading. The Ube2g2:G2BR interac- from ligase domains to reload with ubiquitin (Huang et al., tion also causes an 50-fold increase in affinity 2005; Eletr et al., 2005). Interestingly, there are a few examples between the E2 and RING finger. This results in mark- in which other regions, either within a multisubunit ubiquitin edly increased ubiquitylation by Ube2g2 and the ligase complex or a single subunit E3, bind specific E2s through gp78 RING finger. The significance of this G2BR generally uncharacterized interactions (Madura et al., 1993; Ha- effect is underscored by enhanced ubiquitylation takeyama et al., 1997; Wu et al., 2002; Biederer et al., 1997; Chen observed when Ube2g2 is paired with other RING et al., 2006). This could increase the availability of E2Ub and theoretically allow for reloading of E2 with ubiquitin without finger E3s. These findings uncover a mechanism dissociation from the E3. whereby allosteric effects on an E2 enhance E2- Ubiquitylation and proteasomal degradation perform critical RING finger interactions and, consequently, ubiqui- functions in degradation of misfolded, unassembled, and highly tylation. regulated proteins from the endoplasmic reticulum (ER). ER- associated degradation (ERAD) is a multistep, highly coordi- INTRODUCTION nated process (Nakatsukasa and Brodsky, 2008). In mammals, there are at least five known ER membrane-spanning ERAD Ubiquitylation occurs as the result of a hierarchical multienzyme E3s. Among these is gp78, also known as the human tumor au- process. Ubiquitin-activating enzyme (E1) activates ubiquitin, tocrine motility factor receptor (AMFR). gp78 is implicated in forming a thiolester linkage between the active site Cys of E1 degradation of T cell antigen receptor subunits, regulatory and the C terminus of ubiquitin. Ubiquitin is transferred to the proteins in lipid metabolism (Kostova et al., 2007), CFTRD508 conserved active site Cys of ubiquitin-conjugating enzymes (Morito et al., 2008), and the metastasis suppressor KAI1 (E2), of which there are more than 30 in mammals. E2s bind to (CD82) (Tsai et al., 2007). specific ubiquitin-protein ligases (E3s), which mediate the trans- gp78 has a complex domain structure for a single subunit E3. fer of ubiquitin to primary amines on substrates or to growing In addition to its RING finger, it has at least three more C-terminal chains of ubiquitin (polyubiquitin or multiubiquitin). In many domains in its extended cytoplasmic tail (Figure 1A). Each of cases, the E3s also undergo auto- or self-ubiquitylation. these is implicated in ubiquitylation and degradation of ERAD There are more than 500 E3s in mammals that can be divided substrates. They include a ubiquitin-binding CUE (coupling of into two major classes. The HECT E3s include 30 E3s and are ubiquitin conjugation to ERAD) domain and a C-terminal binding 674 Molecular Cell 34, 674–685, June 26, 2009 ª2009 Elsevier Inc. Molecular Cell gp78 Ube2g2-Binding Region: Structure and Function 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). site for p97. Unique to gp78 is a high-affinity binding site for its that part of the complex is losing some degree of freedom and cognate E2, Ube2g2. This Ube2g2-binding region (G2BR) is is likely folding into a regular structure. required for the function of gp78 in cells (Chen et al., 2006). We now report that the G2BR binds Ube2g2 through an ‘‘Backside Binding’’ to Ube2g2 Induces G2BR Folding extended interface distinct from sites of RING finger and E1 To identify the binding surface between Ube2g2 and G2BR, we binding. This results in subtle changes in the Ube2g2 core that examined the interaction by monitoring the NMR spectra of each are manifested in functional alterations in loading with ubiquitin molecule independently. First, the Ube2g2 preparation was vali- and a marked increase in affinity for the gp78 RING finger, which dated by confirming the resonance assignments for Ube2g2 is reflected in enhanced ubiquitylation. compared to a previous study (Briggman et al., 2005). A secondary structure analysis based on chemical shifts (Wishart RESULTS and Sykes, 1994) combined with distance information from a 15N-edited NOESY-HSQC spectrum confirmed that our The G2BR Is a High-Affinity Binding Site for Ube2g2 Ube2g2 preparation matched the reported crystal structure To evaluate the interaction between the gp78 G2BR (Figure 1A) (Arai et al., 2006). Titration of G2BR into isotopically labeled and Ube2g2, we employed isothermal titration calorimetry (ITC) Ube2g2 (Figure 1B) yielded slow-exchange NMR spectra and (Figure S1 available online). This confirmed the direct, high- indicated a Kd << 1 mM, consistent with the ITC data. An unex- affinity interaction of Ube2g2 with a 27 amino acid G2BR pectedly large number of the Ube2g2 HN-N peaks shifted in peptide. The 1:1 complex of E2 and G2BR has a dissociation the presence of G2BR, requiring reassignment. The 13C-edited constant (Kd)of21(±4)nM(Table 1). The reaction is exothermic, HSQC spectra of the methyl resonances of Ile, Leu, and Val resi- and the significant free energy change (DG=À43.72 kJ molÀ1) dues (data not shown) indicated a localized binding site, which implies a very stable complex. The decrease in entropy reveals correlates with the largest shifts observed in Figure 1B. The Molecular Cell 34, 674–685, June 26, 2009 ª2009 Elsevier Inc. 675 Molecular Cell gp78 Ube2g2-Binding Region: Structure and Function Table 1. Dissociation Constants of E2-E3 Interactions À1 À1 À1 À1 Complex Kd DH (kJ mol ) DS(JK mol T ) Method NMR Exchange Timescale Ube2g2:G2BR 21 (± 4) nM À56.3 (± 0.01) À41.8 (± 0.04) ITC slow Ube2g2:G2BRDN 740 (± 110) nM À9.80 (± 0.40) 83.6 (± 1.43) ITC slow a Ube2g2:G2BRDC 192 (± 22) mM n.d. n.d. NMR fast Ube2g2:G2BRM4-1 55 (± 18) mM n.d. n.d. NMR fast Ube2g2:G2BRM4-2 9.5 (± 4) mM n.d. n.d. NMR fast Ube2g2:gp78-RING 144 (± 10) mM n.d. n.d. NMR fast (Ube2g2:G2BRDN):gp78-RING 29 (± 5) mM n.d. n.d. NMR fast (Ube2g2:G2BR):gp78-RING 3 (± 1) mM n.d. n.d. NMR fast a n.d. = not determined. secondary structure based on chemical shifts of Ube2g2:G2BR conserved secondary structural elements include a1 = T4–L18, was identical to free Ube2g2, and NOESY spectra of b1 = G23–P28, b2 = E36–M42, b3 = V53–S59, b4 = K70–F73, Ube2g2:G2BR exhibited equivalent patterns of connectivities 310 helix = S91–L93, a2 = V116–A128, a3 = V138–D146, and between HN-HN,HN-methyl, and methyl-methyl protons a4 = R148–L163. Strands b1–b4 form an antiparallel b sheet compared to free Ube2g2, implying that the overall structure of (Figures 2A and 2B). A dynamic region in the b4a2 loop is present Ube2g2 did not change significantly. between residues H94 and W110. This corresponds to an acidic The binding surface was determined by chemical shift extension (aa 96–108) that, in mammals, is limited to Ube2g2 and mapping on Ube2g2 and by intermolecular NOESY experiments.
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