Biophysical Studies on Interactions and Assembly of Full-size E3 Ubiquitin Ligase: SUPPRESSOR OF CYTOKINE SIGNALING 2 (SOCS2)-ELONGIN BC-CULLIN 5-RING BOX PROTEIN 2 (RBX2)*

The multisubunit cullin RING E3 ubiquitin ligases (CRLs) target post-translationally modified substrates for ubiquitination and proteasomal degradation. The suppressors of cytokine signaling (SOCS) proteins play important roles in inflammatory processes, diabetes, and cancer and therefore represent attractive targets for therapeutic intervention. The SOCS proteins, among their other functions, serve as substrate receptors of CRL5 complexes. A member of the CRL family, SOCS2-EloBC-Cul5-Rbx2 (CRL5^SOCS2), binds phosphorylated growth hormone receptor as its main substrate. Here, we demonstrate that the components of CRL5^SOCS2 can be specifically pulled from K562 human cell lysates using beads decorated with phosphorylated growth hormone receptor peptides. Subsequently, SOCS2-EloBC and full-length Cul5-Rbx2, recombinantly expressed in Escherichia coli and in Sf21 insect cells, respectively, were used to reconstitute neddylated and unneddylated CRL5^SOCS2 complexes in vitro. Finally, diverse biophysical methods were employed to study the assembly and interactions within the complexes. Unlike other E3 ligases, CRL5^SOCS2 was found to exist in a monomeric state as confirmed by size exclusion chromatography with inline multiangle static light scattering and native MS. Affinities of the protein-protein interactions within the multisubunit complex were measured by isothermal titration calorimetry. A structural model for full-size neddylated and unneddylated CRL5^SOCS2 complexes is supported by traveling wave ion mobility mass spectrometry data.     

The E3 ubiquitin ligase HOIL-1 induces the polyubiquitination and degradation of SOCS6 associated proteins

The suppressor of cytokine signaling (SOCS) proteins are thought to exert their function through the recruitment of interacting-proteins to the ubiquitin/proteasome degradation pathway. All SOCS proteins bind an Elongin BC E3 ubiquitin ligase complex through the common Socs-box. Here, we show that haem-oxidized IRP2 ubiquitin ligase-1 (HOIL-1), another E3 ubiquitin ligase, interacts with SOCS6. The Ubl domain of HOIL-1 and the SH2 and Socs-box domains of SOCS6 are required for the interaction. HOIL-1 expression stabilizes SOCS6 and induces the ubiquitination and degradation of proteins associated with SOCS6. These data suggest that SOCS proteins may interact with different E3 ubiquitin ligases in addition to a common Elongin BC E3 complex.     

The Mechanism of Ubiquitination in the Cullin-RING E3 Ligase Machinery: Conformational Control of Substrate Orientation

In cullin-RING E3 ubiquitin ligases, substrate binding proteins, such as VHL-box, SOCS-box or the F-box proteins, recruit substrates for ubiquitination, accurately positioning and orienting the substrates for ubiquitin transfer. Yet, how the E3 machinery precisely positions the substrate is unknown. Here, we simulated nine substrate binding proteins: Skp2, Fbw7, β-TrCP1, Cdc4, Fbs1, TIR1, pVHL, SOCS2, and SOCS4, in the unbound form and bound to Skp1, ASK1 or Elongin C. All nine proteins have two domains: one binds to the substrate; the other to E3 ligase modules Skp1/ASK1/Elongin C. We discovered that in all cases the flexible inter-domain linker serves as a hinge, rotating the substrate binding domain, optimally and accurately positioning it for ubiquitin transfer. We observed a conserved proline in the linker of all nine proteins. In all cases, the prolines pucker substantially and the pucker is associated with the backbone rotation toward the E2/ubiquitin. We further observed that the linker flexibility could be regulated allosterically by binding events associated with either domain. We conclude that the flexible linker in the substrate binding proteins orients the substrate for the ubiquitin transfer. Our findings provide a mechanism for ubiquitination and polyubiquitination, illustrating that these processes are under conformational control.     

Molecular Insight into the Conformational Dynamics of the Elongin BC Complex and Its Interaction with HIV-1 Vif

The human immunodeficiency virus type 1 virion infectivity factor (Vif) inhibits the innate viral immunity afforded by the APOBEC3 family of cytidine deaminases. Vif targets the APOBEC3 family for poly-ubiquitination and subsequent proteasomal degradation by linking the Elongin-BC-dependent ubiquitin ligase complex with the APOBEC3 proteins. The interaction between Vif and the heterodimeric Elongin BC complex, which is mediated by Vif's viral suppressor of cytokine signaling box, is essential for Vif function. The biophysical consequences of the full-length Vif:Elongin BC interaction have not been extensively reported. In this study, hydrogen exchange mass spectrometry was used to dissect the Vif:Elongin BC interaction. Elongin C was found to be highly dynamic in the Elongin BC complex while Elongin B was much more stable. Recombinant full-length Vif interacted with the Elongin BC complex in vitro with a K_d of 1.9 μM and resulted in observable changes in deuterium uptake in both Elongin C and B. Upon binding to Elongin BC, no significant global conformational changes were detected in Vif by hydrogen exchange mass spectrometry, but a short fragment of Vif that consisted of the viral suppressor of cytokine signaling box showed decreased deuterium incorporation upon Elongin BC incubation, suggesting that this region folds upon binding.     

The SOCS2 ubiquitin ligase complex regulates growth hormone receptor levels

Growth Hormone is essential for the regulation of growth and the homeostatic control of intermediary metabolism. GH actions are mediated by the Growth Hormone Receptor; a member of the cytokine receptor super family that signals chiefly through the JAK2/STAT5 pathway. Target tissue responsiveness to GH is under regulatory control to avoid excessive and off-target effects upon GHR activation. The suppressor of cytokine signalling 2 (SOCS) is a key regulator of GHR sensitivity. This is clearly shown in mice where the SOCS2 gene has been inactivated, which show 30–40% increase in body length, a phenotype that is dependent on endogenous GH secretion. SOCS2 is a GH-stimulated, STAT5b-regulated gene that acts in a negative feedback loop to downregulate GHR signalling. Since the biochemical basis for these actions is poorly understood, we studied the molecular function of SOCS2. We demonstrated that SOCS2 is part of a multimeric complex with intrinsic ubiquitin ligase activity. Mutational analysis shows that the interaction with Elongin B/C controls SOCS2 protein turnover and affects its molecular activity. Increased GHR levels were observed in livers from SOCS2^−/− mice and in the absence of SOCS2 in in vitro experiments. We showed that SOCS2 regulates cellular GHR levels through direct ubiquitination and in a proteasomally dependent manner. We also confirmed the importance of the SOCS-box for the proper function of SOCS2. Finally, we identified two phosphotyrosine residues in the GHR to be responsible for the interaction with SOCS2, but only Y487 to account for the effects of SOCS2. The demonstration that SOCS2 is an ubiquitin ligase for the GHR unveils the molecular basis for its physiological actions.     

Muf1, a novel Elongin BC-interacting leucine-rich repeat protein that can assemble with Cul5 and Rbx1 to reconstitute a ubiquitin ligase

The heterodimeric Elongin BC complex has been shown to interact in vitro and in mammalian cells with a conserved BC-box motif found in a growing number of proteins including RNA polymerase II elongation factor Elongin A, SOCS-box proteins, and the von Hippel-Lindau (VHL) tumor suppressor protein. Recently, the VHL-Elongin BC complex was found to interact with a module composed of Cullin family member Cul2 and RING-H2 finger protein Rbx1 to reconstitute a novel E3 ubiquitin ligase that activates ubiquitylation by the E2 ubiquitin-conjugating enzymes Ubc5 and Cdc34. In the context of the VHL ubiquitin ligase, Elongin BC functions as an adaptor that links the VHL protein to the Cul2/Rbx1 module, raising the possibility that the Elongin BC complex could function as an integral component of a larger family of E3 ubiquitin ligases by linking alternative BC-box proteins to Cullin/Rbx1 modules. In this report, we describe identification and purification from rat liver of a novel leucine-rich repeat-containing BC-box protein, MUF1, which we demonstrate is capable of assembling with a Cullin/Rbx1 module containing the Cullin family member Cul5 to reconstitute ubiquitin ligase activity. In addition, we show that the additional BC-box proteins Elongin A, SOCS1, and WSB1 are also capable of assembling with the Cul5/Rbx1 module to reconstitute potential ubiquitin ligases. Taken together, our findings identify MUF1 as a new member of the BC-box family of proteins, and they predict the existence of a larger family of Elongin BC-based E3 ubiquitin ligases.     

The role of cullin 5-containing ubiquitin ligases

The suppressor of cytokine signaling (SOCS) box consists of the BC box and the cullin 5 (Cul5) box, which interact with Elongin BC and Cul5, respectively. SOCS box-containing proteins have ubiquitin ligase activity mediated by the formation of a complex with the scaffold protein Cul5 and the RING domain protein Rbx2, and are thereby members of the cullin RING ligase superfamily. Cul5-type ubiquitin ligases have a variety of substrates that are targeted for polyubiquitination and proteasomal degradation. Here, we review the current knowledge on the identification of Cul5 and the regulation of its expression, as well as the signaling pathways regulated by Cul5 and how viruses highjack the Cul5 system to overcome antiviral responses.     

The SOCS box of SOCS-1 accelerates ubiquitin-dependent proteolysis of TEL-JAK2

Fusion of the TEL gene on 12p13 to the JAK2 tyrosine kinase gene on 9p24 has been found in human leukemia. TEL-mediated oligomerization of JAK2 results in constitutive activation of the tyrosine kinase (JH1) domain and confers cytokine-independent proliferation on interleukin-3-dependent Ba/F3 cells. Forced expression of the JAK inhibitor gene SOCS1/JAB/SSI-1 induced apoptosis of TEL-JAK2-transformed Ba/F3 cells. This suppression of TEL-JAK2 activity was dependent on SOCS box-mediated proteasomal degradation of TEL-JAK2 rather than on kinase inhibition. Degradation of JAK2 depended on its phosphorylation and its high affinity binding with SOCS1 through the kinase inhibitory region and the SH2 domain. It has been demonstrated that von Hippel-Lindau disease (VHL) tumor-suppressor gene product possesses the SOCS box that forms a complex with Elongin B and C and Cullin-2, and it functions as a ubiquitin ligase. The SOCS box of SOCS1/JAB has also been shown to interact with Elongins; however, ubiquitin ligase activity has not been demonstrated. We found that the SOCS box interacted with Cullin-2 and promoted ubiquitination of TEL-JAK2. Furthermore, overexpression of dominant negative Cullin-2 suppressed SOCS1-dependent TEL-JAK2 degradation. Our study demonstrates the substrate-specific E3 ubiquitin-ligase-like activity of SOCS1 for activated JAK2 and may provide a novel strategy for the suppression of oncogenic tyrosine kinases.     

A mutant form of JAB/SOCS1 augments the cytokine-induced JAK/STAT pathway by accelerating degradation of wild-type JAB/CIS family proteins through the SOCS-box

Cytokines exert biological functions by activating Janus tyrosine kinases (JAKs), and JAK inhibitors JAB (also referred to as SOCS1 and SSI1) and CIS3 (SOCS3) play an essential role in the negative regulation of cytokine signaling. We have found that transgenic (Tg) mice expressing a mutant JAB (F59D-JAB) exhibited a more potent STAT3 activation and a more severe colitis than did wild-type littermates after treatment with dextran sulfate sodium. We now find that there is a prolonged activation of JAKs and STATs in response to a number of cytokines in T cells from Tg mice with lck promoter-driven F59D-JAB. Overexpression of F59D-JAB also sustained activation of JAK2 in Ba/F3 cells. These data suggested that F59D-JAB up-regulated STAT activity by sustaining JAK activation. To elucidate molecular mechanisms related to F59D-JAB, we analyzed the effects of F59D-JAB on the JAK/STAT pathway using the 293 cell transient expression system. We found that the C-terminal SOCS-box played an essential role in augmenting cytokine signaling by F59D-JAB. The SOCS-box interacted with the Elongin BC complex, and this interaction stabilized JAB. F59D-JAB induced destabilization of wild-type JAB, whereas overexpression of Elongin BC canceled this effect. Levels of endogenous JAB and CIS3 in T cells from F59D-JAB Tg-mouse were lower than in wild-type mice. We propose that F59D-JAB destabilizes wild-type, endogenous JAB and CIS3 by chelating the Elongin BC complex, thereby sustaining JAK activation.     

Substrate binding induces a cooperative conformational change in the 12S subunit of transcarboxylase: Raman crystallographic evidence

The 12S subunit of transcarboxylase is a 338 000 Da hexamer that transfers carboxlylate from methylmalonyl-CoA (MM-CoA) to biotin; in turn, the biotin transfers the carboxylate to pyruvate on another subunit, the 5S. Here, Raman difference microscopy is used to study the binding of substrate and product, and their analogues, to single crystals of 12S. A single crystal is the medium of choice because it provides Raman data of unprecedented quality. Crystalline ligand-protein complexes were formed by cocrystallization or by the soaking in/soaking out method. Raman difference spectra were obtained by subtracting the spectrum of the apo crystal from that of a crystal with the substrate or product bound. Raman difference spectra from crystals with the substrate bound are dominated by bands from the protein's amide bonds and aromatic side chain residues. In contrast, Raman difference spectra involving the product, propionyl-CoA, are dominated by modes from the ligand. These results show that substrate binding triggers a conformational change in 12S, whereas product binding does not. The conformational change involves an increase in the amount of alpha-helix since markers for this secondary structure are prominent in the difference spectra of the substrate complex. The number of MM-CoA ligands bound per 12S hexamer can be gauged from the intensity of the MM-CoA Raman features and the fact that the protein concentration in the crystals is known from X-ray crystallographic data. Most crystal samples had six MM-CoAs per hexamer although a few, from different soaking experiments, contained only 1-2. However, both sets of crystals showed the same degree of protein conformational change, indicating that the change induced by the substrate is cooperative. This effect allowed us to record the Raman spectrum of bound MM-CoA without interference from protein modes; the Raman spectrum of a 12S crystal containing 2 MM-CoA ligands per hexamer was subtracted from the Raman spectrum of a 12S crystal containing six MM-CoA ligands per hexamer. The conformational change is reversible and can be controlled by soaking out or soaking in the ligand, using either concentrated ammonium sulfate solutions or the solution used in the crystallization trials. Malonyl-CoA also binds to 12S crystals and brings about conformational changes identical to those seen for MM-CoA; in addition, butyryl-CoA binds and behaves in a manner similar to propionyl-CoA. These data implicate the -COO- group on MM-CoA (that is transferred to biotin in the reaction on the intact enzyme) as the agent bringing about the cooperative conformational change in 12S.     

Rbx1 Flexible Linker Facilitates Cullin-RING Ligase Function Before Neddylation and After Deneddylation

In ubiquitination, cullin-RING E3 ubiquitin ligases (CRLs) assist in ubiquitin transfer from ubiquitin-conjugating enzyme E2 to the substrate. Neddylation, which involves NEDD8 transfer from E2 to E3-cullin, stimulates ubiquitination by inducing conformational change in CRLs. However, deneddylation, which removes NEDD8 from cullin, does not suppress ubiquitination in vivo, raising the question of how neddylation/deneddylation exerts its effects. Using molecular-dynamics simulations, we demonstrate that before neddylation occurs, the linker flexibility of Rbx1, a CRL component, leads to conformational changes in CRLs that allow neddylation and initiation of ubiquitination. These large NEDD8-induced conformational changes are retained after deneddylation, allowing both initiation of the ubiquitination process and ubiquitin chain elongation after deneddylation. Furthermore, mutation of lysine, the cullin residue to which NEDD8 covalently attaches, dramatically reduces CRL conformational changes, suggesting that the acceptor lysine allosterically regulates CRLs. Thus, our results imply that neddylation stimulates ubiquitination by CRL conformational control via lysine modification.     

The SOCS box domain of SOCS3: structure and interaction with the elonginBC-cullin5 ubiquitin ligase

Suppressor of cytokine signalling 3 (SOCS3) is responsible for regulating the cellular response to a variety of cytokines, including interleukin 6 and leukaemia inhibitory factor. Identification of the SOCS box domain led to the hypothesis that SOCS3 can associate with functional E3 ubiquitin ligases and thereby induce the degradation of bound signalling proteins. This model relies upon an interaction between the SOCS box, elonginBC and a cullin protein that forms the E3 ligase scaffold. We have investigated this interaction in vitro using purified components and show that SOCS3 binds to elonginBC and cullin5 with high affinity. The SOCS3-elonginBC interaction was further characterised by determining the solution structure of the SOCS box-elonginBC ternary complex and by deletion and alanine scanning mutagenesis of the SOCS box. These studies revealed that conformational flexibility is a key feature of the SOCS-elonginBC interaction. In particular, the SOCS box is disordered in isolation and only becomes structured upon elonginBC association. The interaction depends upon the first 12 residues of the SOCS box domain and particularly on a deeply buried, conserved leucine. The SOCS box, when bound to elonginBC, binds tightly to cullin5 with 100 nM affinity. Domains upstream of the SOCS box are not required for elonginBC or cullin5 association, indicating that the SOCS box acts as an independent binding domain capable of recruiting elonginBC and cullin5 to promote E3 ligase formation.     

Crystal structure of the reduced form of p-hydroxybenzoate hydroxylase refined at 2.3 A resolution.

The crystal structure of the reduced form of the enzyme p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, complexed with its substrate p-hydroxybenzoate, has been obtained by protein X-ray crystallography. Crystals of the reduced form were prepared by soaking crystals of the oxidized enzyme-substrate complex in deaerated mother liquor containing 300-400 mM NADPH. A rapid bleaching of the crystals indicated the reduction of the enzyme-bound FAD by NADPH. This was confirmed by single crystal spectroscopy. X-ray data to 2.3 A were collected on oscillation films using a rotating anode generator as an X-ray source. After data processing and reduction, restrained least squares refinement using the 1.9 A structure of the oxidized enzyme-substrate complex as a starting model, yielded a crystallographic R-factor of 14.8% for 11,394 reflections. The final model of the reduced complex contains 3,098 protein atoms, the FAD molecule, the substrate p-hydroxybenzoate and 322 solvent molecules. The structures of the oxidized and reduced forms of the enzyme-substrate complex were found to be very similar. The root-mean-square discrepancy for all atoms between both structures was 0.38 A. The flavin ring is almost completely planar in the final model, although it was allowed to bend or twist during refinement. The observed angle between the benzene and the pyrimidine ring is 2 degrees. This value should be compared with observed values of 10 degrees for the oxidized enzyme-substrate complex and 19 degrees for the enzyme-product complex. The position of the substrate is virtually unaltered with respect to its position in the oxidized enzyme. No trace of a bound NADP+ or NADPH molecule was found.