Structure of the MID1 tandem B-boxes reveals an interaction reminiscent of intermolecular ring heterodimers

The tripartite motif (TRIM) protein family, defined by N-terminal RING, B-box, and coiled-coil (RBCC) domains, consists of either a single type 2 B-box domain or tandem B-box domains of type 1 and type 2 (B1B2). Here, we report the first structure of the B-box domains in their native tandem orientation. The B-boxes are from Midline-1, a putative ubiquitin E3 ligase that is required for the proteosomal degradation of the catalytic subunit of protein phosphatase 2A (PP2Ac). This function of MID1 is facilitated by the direct binding of Alpha4, a regulatory subunit of PP2Ac, to B-box1, while B-box2 appears to influence this interaction. Both B-box1 and B-box2 bind two zinc atoms in a cross-brace motif and adopt a similar betabetaalpha structure reminiscent of the RING, PHD, ZZ, and U-box domains, although they differ from each other and with RING domains in the spacing of their zinc-binding residues. The two B-box domains pack against each other with the interface formed by residues located on the structured loop consisting of the two antiparallel beta-strands. The surface area of the interface is 188 A2 (17% of the total surface). Consistent with the globular structure, the Tm of the tandem B-box domain (59 degrees C) is higher than the individual domains, supporting a stable interaction between the B-box 1 and 2 domains. Notably, the interaction is reminiscent of the interaction of recently determined RING dimers, suggesting the possibility of an evolutionarily conserved role for B-box2 domains in regulating functional RING-type folds.     

Solution structure of the MID1 B-box2 CHC(D/C)C(2)H(2) zinc-binding domain: insights into an evolutionarily conserved RING fold

The B-box type 2 domain is a prominent feature of a large and growing family of RING, B-box, coiled-coil (RBCC) domain-containing proteins and is also present in more than 1500 additional proteins. Most proteins usually contain a single B-box2 domain, although some proteins contain tandem domains consisting of both type 1 and type 2 B-boxes, which actually share little sequence similarity. Recently, we determined the solution structure of B-box1 from MID1, a putative E3 ubiquitin ligase that is mutated in X-linked Opitz G/BBB syndrome, and showed that it adopted a betabetaalpha RING-like fold. Here, we report the tertiary structure of the B-box2 (CHC(D/C)C(2)H(2)) domain from MID1 using multidimensional NMR spectroscopy. This MID1 B-box2 domain consists of a short alpha-helix and a structured loop with two short anti-parallel beta-strands and adopts a tertiary structure similar to the B-box1 and RING structures, even though there is minimal primary sequence similarity between these domains. By mutagenesis, ESI-FTICR and ICP mass spectrometry, we show that the B-box2 domain coordinates two zinc atoms with a 'cross-brace' pattern: one by Cys175, His178, Cys195 and Cys198 and the other by Cys187, Asp190, His204, and His207. Interestingly, this is the first case that an aspartic acid is involved in zinc atom coordination in a zinc-finger domain, although aspartic acid has been shown to coordinate non-catalytic zinc in matrix metalloproteinases. In addition, the finding of a Cys195Phe substitution identified in a patient with X-linked Opitz GBBB syndrome supports the importance of proper zinc coordination for the function of the MID1 B-box2 domain. Notably, however, our structure differs from the only other published B-box2 structure, that from XNF7, which was shown to coordinate one zinc atom. Finally, the similarity in tertiary structures of the B-box2, B-box1 and RING domains suggests these domains have evolved from a common ancestor.     

TRIM16 acts as an E3 ubiquitin ligase and can heterodimerize with other TRIM family members

The TRIM family of proteins is distinguished by its tripartite motif (TRIM). Typically, TRIM proteins contain a RING finger domain, one or two B-box domains, a coiled-coil domain and the more variable C-terminal domains. TRIM16 does not have a RING domain but does harbour two B-box domains. Here we showed that TRIM16 homodimerized through its coiled-coil domain and heterodimerized with other TRIM family members; TRIM24, Promyelocytic leukaemia (PML) protein and Midline-1 (MID1). Although, TRIM16 has no classic RING domain, three-dimensional modelling of TRIM16 suggested that its B-box domains adopts RING-like folds leading to the hypothesis that TRIM16 acts as an ubiquitin ligase. Consistent with this hypothesis, we demonstrated that TRIM16, devoid of a classical RING domain had auto-polyubiquitination activity and acted as an E3 ubiquitin ligase in vivo and in vitro assays. Thus via its unique structure, TRIM16 possesses both heterodimerization function with other TRIM proteins and also has E3 ubiquitin ligase activity.     

BTBD1 and BTBD2 colocalize to cytoplasmic bodies with the RBCC/tripartite motif protein,TRIM5delta

We previously identified BTBD1 and BTBD2 as novel topoisomerase I-interacting proteins that share 80% amino acid identity. Here we report the characterization of their subcellular localization. In a number of mouse and human cells, BTBD1 and BTBD2 (BTBD1/2) colocalized to punctate or elongated cytoplasmic bodies (< 5 microm long and several per cell) that were larger and more elongated in cancer cell lines than in fibroblasts and myoblasts. A search for potential colocalizing proteins identified TRIM family members that localize to morphologically similar cytoplasmic bodies, which were then tested for colocalization with BTBD1/2. TRIM5delta, expressed as a GFP fusion, colocalized with BTBD1/2 immunostaining and appeared to serve as a scaffold for the assembly of endogenous BTBD1/2 proteins. TRIM family members contain a RING domain, B-box(es), and coiled-coil regions, which have a characteristic order and spacing (RBCC domain). RING-dependent ubiquitin ligase activity and multimerization via the coiled-coil region may be defining properties of the RBCC/TRIM protein family. We found that TRIM5delta with a deleted coiled-coil region or a mutated RING domain failed to colocalize with BTBD1/2. Additionally, TRIM5delta ubiquitylated itself in a RING finger- and UbcH5B-dependent manner. BTBD1/2 each contain a PHR-similarity region, repeated twice on the putative ubiquitin ligases PAM, highwire and RPM-1, which also contain a RING and B-box. Thus, four protein modules found on each of these putative ubiquitin ligases, a RING, a B-box and two PHR repeats, are present on BTBD1/2 and TRIM5delta that are colocalized to cytoplasmic bodies.     

TRIM/RBCC, a novel class of 'single protein RING finger' E3 ubiquitin ligases

The TRIM/RBCC proteins are defined by the presence of the tripartite motif composed of a RING domain, one or two B-box motifs and a coiled-coil region. These proteins are involved in a plethora of cellular processes such as apoptosis, cell cycle regulation and viral response. Consistently, their alteration results in many diverse pathological conditions. The highly conserved modular structure of these proteins suggests that a common biochemical function may underlie their assorted cellular roles. Here, we review recent data indicating that some TRIM/RBCC proteins are implicated in ubiquitination and propose that this large protein family represents a novel class of 'single protein RING finger' ubiquitin E3 ligases.     

Structure and catalytic activation of the TRIM23 RING E3 ubiquitin ligase

Tripartite motif (TRIM) proteins comprise a large family of RING‐type ubiquitin E3 ligases that regulate important biological processes. An emerging general model is that TRIMs form elongated antiparallel coiled‐coil dimers that prevent interaction of the two attendant RING domains. The RING domains themselves bind E2 conjugating enzymes as dimers, implying that an active TRIM ligase requires higher‐order oligomerization of the basal coiled‐coil dimers. Here, we report crystal structures of the TRIM23 RING domain in isolation and in complex with an E2–ubiquitin conjugate. Our results indicate that TRIM23 enzymatic activity requires RING dimerization, consistent with the general model of TRIM activation.     

Origin and Diversification of TRIM Ubiquitin Ligases

Most proteins of the TRIM family (also known as RBCC family) are ubiquitin ligases that share a peculiar protein structure, characterized by including an N-terminal RING finger domain closely followed by one or two B-boxes. Additional protein domains found at their C termini have been used to classify TRIM proteins into classes. TRIMs are involved in multiple cellular processes and many of them are essential components of the innate immunity system of animal species. In humans, it has been shown that mutations in several TRIM-encoding genes lead to diverse genetic diseases and contribute to several types of cancer. They had been hitherto detected only in animals. In this work, by comprehensively analyzing the available diversity of TRIM and TRIM-like protein sequences and evaluating their evolutionary patterns, an improved classification of the TRIM family is obtained. Members of one of the TRIM subfamilies defined, called Subfamily A, turn to be present not only in animals, but also in many other eukaryotes, such as fungi, apusozoans, alveolates, excavates and plants. The rest of subfamilies are animal-specific and several of them originated only recently. Subfamily A proteins are characterized by containing a MATH domain, suggesting a potential evolutionary connection between TRIM proteins and a different type of ubiquitin ligases, known as TRAFs, which contain quite similar MATH domains. These results indicate that the TRIM family emerged much earlier than so far thought and contribute to our understanding of its origin and diversification. The structural and evolutionary links with the TRAF family of ubiquitin ligases can be experimentally explored to determine whether functional connections also exist.     

The contribution of RING and B-box 2 domains to retroviral restriction mediated by monkey TRIM5alpha

TRIM5alpha is a cytoplasmic protein that mediates a post-entry block to infection by some retroviruses. TRIM5alpha contains a tripartite motif (TRIM), which includes RING, B-box 2, and coiled-coil domains, and a C-terminal B30.2 (SPRY) domain. We investigated the contribution of the RING and B-box 2 domains to the antiretroviral activity of rhesus monkey TRIM5alpha (TRIM5alpharh), which potently restricts infection by human immunodeficiency virus, type 1 (HIV-1) and simian immunodeficiency virus of African green monkeys (SIVagm). Disruption of the RING domain caused mislocalization of TRIM5alpharh so that the cytoplasmic level of the protein was decreased compared with that of the wild-type protein. Nonetheless, partial ability to restrict HIV-1 and SIVagm was retained by the RING domain mutants. By contrast, although TRIM5alpharh mutants with disrupted B-box 2 domains were efficiently expressed and correctly localized to the cytoplasm, antiretroviral activity was absent. The B-box 2 mutants colocalized and associated with wild-type TRIM5alpharh and exerted dominant-negative effects on the antiretroviral activity of the wild-type protein. Taken together with other data, these results indicate that functionally defective TRIM5alpharh molecules that retain a coiled coil can act as dominant-negative inhibitors of wild-type TRIM5alpharh function. The RING domain of TRIM5alpharh is not absolutely required for retrovirus restriction but can influence cytoplasmic levels of the protein and thus indirectly alter function. The B-box 2 domain, by contrast, appears to be essential for efficient retrovirus restriction.     

Functional replacement of the RING, B-box 2, and coiled-coil domains of tripartite motif 5alpha (TRIM5alpha) by heterologous TRIM domains

Tripartite motif 5alpha (TRIM5alpha) restricts some retroviruses, including human immunodeficiency virus type 1 (HIV-1), from infecting the cells of particular species. TRIM5alpha is a member of the TRIM family of proteins, which contain RING, B-box, coiled-coil (CC), and, in some cases, B30.2(SPRY) domains. Here we investigated the abilities of domains from TRIM proteins (TRIM6, TRIM34, and TRIM21) that do not restrict HIV-1 infection to substitute for the domains of rhesus monkey TRIM5alpha (TRIM5alpha(rh)). The RING, B-box 2, and CC domains of the paralogous TRIM6 and TRIM34 proteins functionally replaced the corresponding TRIM5alpha(rh) domains, allowing HIV-1 restriction. By contrast, similar chimeras containing the components of TRIM21, a slightly more distant relative of TRIM5, did not restrict HIV-1 infection. The TRIM21 B-box 2 domain and its flanking linker regions contributed to the functional defectiveness of these chimeras. All of the chimeric proteins formed trimers. All of the chimeras that restricted HIV-1 infection bound the assembled HIV-1 capsid complexes. These results indicate that heterologous RING, B-box 2, and CC domains from related TRIM proteins can functionally substitute for TRIM5alpha(rh) domains.     

Functional interactions between ubiquitin E2 enzymes and TRIM proteins

The TRIM (tripartite motif) family of proteins is characterized by the presence of the tripartite motif module, composed of a RING domain, one or two B-box domains and a coiled-coil region. TRIM proteins are involved in many cellular processes and represent the largest subfamily of RING-containing putative ubiquitin E3 ligases. Whereas their role as E3 ubiquitin ligases has been presumed, and in several cases established, little is known about their specific interactions with the ubiquitin-conjugating E2 enzymes or UBE2s. In the present paper, we report a thorough screening of interactions between the TRIM and UBE2 families. We found a general preference of the TRIM proteins for the D and E classes of UBE2 enzymes, but we also revealed very specific interactions between TRIM9 and UBE2G2, and TRIM32 and UBE2V1/2. Furthermore, we demonstrated that the TRIM E3 activity is only manifest with the UBE2 with which they interact. For most specific interactions, we could also observe subcellular co-localization of the TRIM involved and its cognate UBE2 enzyme, suggesting that the specific selection of TRIM-UBE2 pairs has physiological relevance. Our findings represent the basis for future studies on the specific reactions catalysed by the TRIM E3 ligases to determine the fate of their targets.     

Structure of the HHARI Catalytic Domain Shows Glimpses of a HECT E3 Ligase

The ubiquitin-signaling pathway utilizes E1 activating, E2 conjugating, and E3 ligase enzymes to sequentially transfer the small modifier protein ubiquitin to a substrate protein. During the last step of this cascade different types of E3 ligases either act as scaffolds to recruit an E2 enzyme and substrate (RING), or form an ubiquitin-thioester intermediate prior to transferring ubiquitin to a substrate (HECT). The RING-inBetweenRING-RING (RBR) proteins constitute a unique group of E3 ubiquitin ligases that includes the Human Homologue of Drosophila Ariadne (HHARI). These E3 ligases are proposed to use a hybrid RING/HECT mechanism whereby the enzyme uses facets of both the RING and HECT enzymes to transfer ubiquitin to a substrate. We now present the solution structure of the HHARI RING2 domain, the key portion of this E3 ligase required for the RING/HECT hybrid mechanism. The structure shows the domain possesses two Zn²⁺-binding sites and a single exposed cysteine used for ubiquitin catalysis. A structural comparison of the RING2 domain with the HECT E3 ligase NEDD4 reveals a near mirror image of the cysteine and histidine residues in the catalytic site. Further, a tandem pair of aromatic residues exists near the C-terminus of the HHARI RING2 domain that is conserved in other RBR E3 ligases. One of these aromatic residues is remotely located from the catalytic site that is reminiscent of the location found in HECT E3 enzymes where it is used for ubiquitin catalysis. These observations provide an initial structural rationale for the RING/HECT hybrid mechanism for ubiquitination used by the RBR E3 ligases.     

Structure-based mutagenesis of the substrate-recognition domain of Nrdp1/FLRF identifies the binding site for the receptor tyrosine kinase ErbB3

The E3 ubiquitin ligase neuregulin receptor degrading protein 1 (Nrdp1) mediates the ligand-independent degradation of the epidermal growth factor receptor family member ErbB3/HER3. By regulating cellular levels of ErbB3, Nrdp1 influences ErbB3-mediated signaling, which is essential for normal vertebrate development. Nrdp1 belongs to the tripartite or RBCC (RING, B-box, coiled-coil) family of ubiquitin ligases in which the RING domain is responsible for ubiquitin ligation and a variable C-terminal region mediates substrate recognition. We report here the 1.95 A crystal structure of the C-terminal domain of Nrdp1 and show that this domain is sufficient to mediate ErbB3 binding. Furthermore, we have used site-directed mutagenesis to map regions of the Nrdp1 surface that are important for interacting with ErbB3 and mediating its degradation in transfected cells. The ErbB3-binding site localizes to a region of Nrdp1 that is conserved from invertebrates to vertebrates, in contrast to ErbB3, which is only found in vertebrates. This observation suggests that Nrdp1 uses a common binding site to recognize its targets in different species.     

UHRF2, a Ubiquitin E3 Ligase,Acts as a Small Ubiquitin-like Modifier E3 Ligase for Zinc Finger Protein 131

Small ubiquitin-like modifier (SUMO), a member of the ubiquitin-related protein family, is covalently conjugated to lysine residues of its substrates in a process referred to as SUMOylation. SUMOylation occurs through a series of enzymatic reactions analogous to that of the ubiquitination pathway, resulting in modification of the biochemical and functional properties of substrates. To date, four mammalian SUMO isoforms, a single heterodimeric SUMO-activating E1 enzyme SAE1/SAE2, a single SUMO-conjugating E2 enzyme ubiquitin-conjugating enzyme E2I (UBC9), and a few subgroups of SUMO E3 ligases have been identified. Several SUMO E3 ligases such as topoisomerase I binding, arginine/serine-rich (TOPORS), TNF receptor-associated factor 7 (TRAF7), and tripartite motif containing 27 (TRIM27) have dual functions as ubiquitin E3 ligases. Here, we demonstrate that the ubiquitin E3 ligase UHRF2 also acts as a SUMO E3 ligase. UHRF2 effectively enhances zinc finger protein 131 (ZNF131) SUMOylation but does not enhance ZNF131 ubiquitination. In addition, the SUMO E3 activity of UHRF2 on ZNF131 depends on the presence of SET and RING finger-associated and nuclear localization signal-containing region domains, whereas the critical ubiquitin E3 activity RING domain is dispensable. Our findings suggest that UHRF2 has independent functional domains and regulatory mechanisms for these two distinct enzymatic activities.     

Molecular insights into RBR E3 ligase ubiquitin transfer mechanisms

RING‐in‐between‐RING (RBR) ubiquitin (Ub) ligases are a distinct class of E3s, defined by a RING1 domain that binds E2 Ub‐conjugating enzyme and a RING2 domain that contains an active site cysteine similar to HECT‐type E3s. Proposed to function as RING/HECT hybrids, details regarding the Ub transfer mechanism used by RBRs have yet to be defined. When paired with RING‐type E3s, E2s perform the final step of Ub ligation to a substrate. In contrast, when paired with RBR E3s, E2s must transfer Ub onto the E3 to generate a E3~Ub intermediate. We show that RBRs utilize two strategies to ensure transfer of Ub from the E2 onto the E3 active site. First, RING1 domains of HHARI and RNF144 promote open E2~Ubs. Second, we identify a Ub‐binding site on HHARI RING2 important for its recruitment to RING1‐bound E2~Ub. Mutations that ablate Ub binding to HHARI RING2 also decrease RBR ligase activity, consistent with RING2 recruitment being a critical step for the RBR Ub transfer mechanism. Finally, we demonstrate that the mechanism defined here is utilized by a variety of RBRs.     

Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity

TRIM E3 ubiquitin ligases regulate a wide variety of cellular processes and are particularly important during innate immune signalling events. They are characterized by a conserved tripartite motif in their N‐terminal portion which comprises a canonical RING domain, one or two B‐box domains and a coiled‐coil region that mediates ligase dimerization. Self‐association via the coiled‐coil has been suggested to be crucial for catalytic activity of TRIMs; however, the precise molecular mechanism underlying this observation remains elusive. Here, we provide a detailed characterization of the TRIM ligases TRIM25 and TRIM32 and show how their oligomeric state is linked to catalytic activity. The crystal structure of a complex between the TRIM25 RING domain and an ubiquitin‐loaded E2 identifies the structural and mechanistic features that promote a closed E2~Ub conformation to activate the thioester for ubiquitin transfer allowing us to propose a model for the regulation of activity in the full‐length protein. Our data reveal an unexpected diversity in the self‐association mechanism of TRIMs that might be crucial for their biological function.     

FYVE domain targets Pib1p ubiquitin ligase to endosome and vacuolar membranes

Signaling by phosphatidylinositol 3-kinases (PI3Ks) is often mediated by proteins which bind PI3K products directly and are localized to intracellular membranes rich in PI3K products. The FYVE finger domain binds with high specificity to PtdIns3P and proteins containing this domain have been shown to be important components of diverse PI3K signaling pathways. The genome of the yeast Saccharomyces cerevisiae encodes five proteins containing FYVE domains, including Pib1p, whose function is unknown. In addition to a FYVE finger motif, the primary structure of Pib1p contains a region rich in cysteine and histidine residues that we demonstrate binds 2 mol eq of zinc, consistent with this region containing a RING structural domain. The Pib1p RING domain exhibited E2-dependent ubiquitin ligase activity in vitro, indicating that Pib1p is an E3 RING-type ubiquitin ligase. Fluorescence microscopy was used to demonstrate that a GFP-Pib1p fusion protein localized to endosomal and vacuolar membranes and deletional analysis of Pib1p domains indicated that localization of GFP-Pib1p is mediated solely by the FYVE domain. These results suggest that Pib1p mediates ubiquitination of a subset of cellular proteins localized to endosome and vacuolar membranes, and they expand the repertoire of PI3K-regulated pathways identified in eukaryotic cells.     

LUBAC synthesizes linear ubiquitin chains via a thioester intermediate

The linear ubiquitin chain assembly complex (LUBAC) is a RING E3 ligase that regulates immune and inflammatory signalling pathways. Unlike classical RING E3 ligases, LUBAC determines the type of ubiquitin chain being formed, an activity normally associated with the E2 enzyme. We show that the RING-in-between-RING (RBR)-containing region of HOIP-the catalytic subunit of LUBAC-is sufficient to generate linear ubiquitin chains. However, this activity is inhibited by the N-terminal portion of the molecule, an inhibition that is released upon complex formation with HOIL-1L or SHARPIN. Furthermore, we demonstrate that HOIP transfers ubiquitin to the substrate through a thioester intermediate formed by a conserved cysteine in the RING2 domain, supporting the notion that RBR ligases act as RING/HECT hybrids.     

The RBCC gene RFP2 (Leu5) encodes a novel transmembrane E3 ubiquitin ligase involved in ERAD

RFP2, a gene frequently lost in various malignancies, encodes a protein with RING finger, B-box, and coiled-coil domains that belongs to the RBCC/TRIM family of proteins. Here we demonstrate that Rfp2 is an unstable protein with auto-polyubiquitination activity in vivo and in vitro, implying that Rfp2 acts as a RING E3 ubiquitin ligase. Consequently, Rfp2 ubiquitin ligase activity is dependent on an intact RING domain, as RING deficient mutants fail to drive polyubiquitination in vitro and are stabilized in vivo. Immunopurification and tandem mass spectrometry enabled the identification of several putative Rfp2 interacting proteins localized to the endoplasmic reticulum (ER), including valosin-containing protein (VCP), a protein indispensable for ER-associated degradation (ERAD). Importantly, we also show that Rfp2 regulates the degradation of the known ER proteolytic substrate CD3-delta, but not the N-end rule substrate Ub-R-YFP (yellow fluorescent protein), establishing Rfp2 as a novel E3 ligase involved in ERAD. Finally, we show that Rfp2 contains a C-terminal transmembrane domain indispensable for its localization to the ER and that Rfp2 colocalizes with several ER-resident proteins as analyzed by high-resolution immunostaining. In summary, these data are all consistent with a function for Rfp2 as an ERAD E3 ubiquitin ligase.