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Biochemistry. Author manuscript; available in PMC Mar 1, 2012.
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PMCID: PMC3072279
NIHMSID: NIHMS266458

The Cullin-RING E3 ubiquitin ligase CRL4-DCAF1 complex dimerizes via a short helical region in DCAF1

Abstract

The cullin4A-RING E3 ubiquitin ligase (CRL4) is a multisubunit protein complex, comprising cullin4A (CUL4), RING H2 finger protein (RBX1) and DNA damage-binding protein 1 (DDB1). Proteins that recruit specific targets to CRL4 for ubiquitination (ubiquitylation), bind the DDB1 adaptor protein via WD40 domains. Such CRL4 substrate recognition modules are DDB1- and CUL4-associated factors (DCAFs). Here we show that for DCAF1, oligomerization of the protein and the CRL4 complex occurs via a short helical region (residues 845–873) N-terminal to DACF1’s own WD40 domain. This sequence was previously designated as a LIS1 homology (LisH) motif. The oligomerization helix contains a stretch of four Leu residues, which appear to be essential for alpha-helical structure and oligomerization. In vitro reconstituted CRL4-DCAF1 complexes (CRL4DCAF1) form symmetric dimers as visualized by electron microscopy (EM) and dimeric CRL4DCAF1 is a better E3 ligase for in vitro ubiquitination of the UNG2 substrate compared to a monomeric complex.

Protein degradation is a highly regulated process carried out by the ubiquitin-proteasome system (13). It involves covalent modification of protein substrates with multiple copies of ubiquitin (Ub) chains, followed by proteolysis of the Ub-tagged proteins by the 26S proteasome (4, 5). Post-translational ubiquitination is extremely diverse, variable in multimer length and linkage type. While attachment of a single Ub module affects protein localization (6), poly-ubiquitination at different Lys residues of Ub targets proteins for degradation or regulation of activity (79). Ub-modification involves a concerted series of enzymatic reactions. The first enzyme, E1, activates Ub in an Mg-ATP dependent manner, creating a covalent thiolester-linked E1~Ub complex (Ub(T)) with a second Ub bound at the adenylation active site (Ub(A)). Subsequently, the thiolester-linked Ub(T) is transferred to a reactive Cys on a cognate E2 conjugating enzyme by trans-esterification, and Ub-loaded E2s are released from E1s for substrate attachment by E3s (1012). Two different classes of E3 ligases have been characterized that either contain a HECT (homologous to E6-AP C-terminus) or a RING (Really Interesting New Gene) domain. The RING domain protein, RBX1 forms modular complexes with several homologous cullin scaffold subunits (CUL1 to CUL7; reviewed in (13)). Each CUL-RBX1 heterodimer uses a specific adaptor protein to form a multi-subunit complex, referred to as a cullin-RING ubiquitin ligase (CRL1–7). The RING domain mainly functions as a docking site for the E2 enzyme (1416) at the C-terminus of the cullin, while the adaptor proteins bind to the N-terminus of the cullin and position substrate receptors and target proteins for ubiquitination (13, 17, 18). One of the adaptor proteins is DDB1 (DNA damage-binding protein 1), a 127 kDa modular protein that contains three seven-bladed β-propeller WD40 domains (1921). DDB1 specifically associates with CUL4 and binds more than 50 different DCAFs (DDB1-and CUL4-associated factors) that function as substrate receptor proteins (2225). DCAF proteins also contain a WD40 domain that is used for interacting with DDB1(21, 2426).

For most DCAF proteins, neither cellular function nor targets have been identified. Those cellular targets that have been identified were shown to function in chromatin re-modeling, DNA replication, cell cycle regulation, and apoptosis (27, 28). Intriguingly, Vpr, one of the four accessory proteins encoded by HIV-1, interacts with DCAF1, possibly targeting unidentified cellular factors for degradation, and mediating Vpr’s biological activity in G2/M-phase cell-cycle arrest (2937)

It has been argued that in CRL-substrate receptor complexes, differences in substrates sizes may present a problem for poly-ubiquitination given the variable distance between the catalytic site on E2 and the target Lys residue(s) on the substrate. If present, structural constraints imposed by the rigid cullin scaffold in CRLs can be resolved by positioning a flexible hinge somewhere in the complex. This appears to be the achieved by NEDD8 modification at the C-terminus of CUL, shown to impart conformational flexibility onto CUL and RBX1, thereby facilitating poly-ubiquitination of the substrates (38, 39). An alternative mechanism could exploit dimerization and, indeed, for several CRLs, dimerization via the substrate receptor proteins has been reported (reviewed in (18)). Dimerization allows for poly-ubiquitination in trans as well as in cis without affecting the substrate affinity (4044).

Here, we report that DCAF1 can oligomerize through an alpha helical region that was originally identified as a LisH motif. This region is located N-terminal to the WD40 domain, although quite distant with ca. 200 residues separating the two regions. In vitro biochemical studies and electron microscopy of CRL4DCAF1 complexes reveal that the E3 ubiquitin ligase indeed forms an oligomeric supramolecular complex that could aid to accommodate a variety of substrate sizes for ubiquitination.

Experimental Procedures

Cloning and Construction of Plasmids

The cDNA for DCAF1 was purchased from Open Biosystems and codes for residues 97–1507. DCAF1 constructs comprising amino acids 572–1507, 817–1507, 876–1396, 987–1396, and 1005–1507 were cloned into the pIZ/V5-His vectors (Invitrogen), resulting in His6- and V5-tags at the C-termini of the protein constructs. In addition, N-terminally FLAG-tagged or Myc-tagged DCAF1 constructs, encoding residues 96–1507 were cloned into pIZ/V5-His vectors, respectively. Site-directed mutagenesis was carried out on pIZ/V5-His DCAF1 817–1507 using QuickChange kits (Stratagene) to create the L850E/L851E and L852E/L853 mutants. Constructs coding for residues 809–876 were amplified from the pIZ/V5-His DCAF1 817–1507 WT DNA and the two mutants, and cloned into pET32 vectors (Invitrogen). Other DCAF1 constructs coding for residues 809–902 and 841–883 were also cloned into the pET32 vectors. The pET32 vectors were modified to contain a TEV protease recognition site between the thioredoxin fusion partner and the target protein (45). For baculoviruses expressing DCAF1 proteins, constructs coding for 817–1507, 987–1396 and 1005–1507 were cloned into the pENT vector (Invitrogen). Recombinant baculoviruses expressing C-terminally His6-tagged DCAF1 proteins were prepared using Baculodirect C-term (Invitrogen) and pENT-DCAF1 vectors, according to the manufacturer’s protocol. Nucleotide sequences were verified for the entire coding regions of all constructs.

Expression and Purification of Proteins

Thioredoxin(pET32)-tagged DCAF1 809–876 WT, L850E/L851E, L852E/L853E mutant, DCAF1 809–902, and 841–883 constructs containing His6- tag between thioredoxin and the TEV protease recognition site were expressed in E. coli Rosetta 2 (DE3), cultured in Luria-Bertani media, using 0.4 mM IPTG for induction and growth at 18 ºC for 16 h. The DCAF1 809–876 WT, L850E/L851E, L852E/L853E mutant constructs contain an additional C-terminal His6-tag. Uniform 13C/15N labeling of DCAF1 809–876 WT, DCAF1 809– 902, and DCAF1 841–883 proteins was carried out by growth in modified medium using 15NH4Cl and/or 13C6-glucose as sole nitrogen and carbon sources, respectively. Soluble forms of His6-tagged proteins were purified using 5 mL Ni-NTA columns (GE Heatlthcare) and aggregated materials were removed by gel-filtration column chromatography using Hi-Load Superdex200 16/60 column (GE Healthcare), equilibrated with a buffer containing 25 mM sodium phosphate, pH 7.5, 150 mM NaCl, 1 mM DTT, 10% glycerol, and 0.02% sodium azide. Thioredoxin-tagged DCAF1 proteins were digested with TEV protease, and DCAF1 proteins were purified over an 8 mL MONO Q column (GE Healthcare), equilibrated with 25 mM TrisHCl, pH 8.5, and 0.02% azide, using a 0–1 M NaCl gradient for elution. C-terminally His6- tagged DCAF1 817–1507, 1005–1507, and 987–1396 proteins were co-expressed with N-terminally His10- and FLAG-tagged DDB1 in SF21 insect cells (Invitrogen). The N-terminally His6-tagged RBX1 and CUL4A were also co-expressed in SF21 cells. Expression of proteins in insect cells and purification were performed as described previously (46). For preparation of multi-protein complexes composed of CUL4A, RBX1, DDB1, and DCAF1 proteins, all components were mixed at equimolar ratios and the protein complexes were purified over an 8 mL MONO Q column (GE Healthcare) at pH 7.5, using a 0–1 M NaCl gradient for elution. Purified complexes were analyzed by SDS-PAGE and native PAGE and visualized by Coomassie Blue staining. All other proteins used in this report were prepared as previously described (46).

Insect Cell Cultures and Transient Transfection

SF9 cells (Invitrogen) were cultured in SF-900 II SFM medium containing 2% fetal bovine serum. Cells were plated in 10 cm plates at 90% confluency 2 h prior to transient transfection. The cells were co-transfected with a total of 16 μg pIZ/V5-His plasmids coding for the various DCAF1 constructs as indicated using Cellfectin II (Invitrogen), according to the manufacturer’s protocol. Cells were harvested 48 h after transfection and subjected to immunoprecipitation as described below. Immunoprecipitation and Immunoblotting. Transiently transfected SF9 cells were harvested and treated with 400 μL of a lysis buffer containing phosphate buffered saline (PBS), 5% glycerol, 1% Tween 20, 1% NP-40, 0.2 mM phenylmethylsulfony fluoride, and protease inhibitor cocktail (EMD Biosciences). The lysates were incubated with 40 μL of anti-FLAG agarose affinity gel for 4 h, and the beads were washed four times with 500 μL of the lysis buffer. Immunoprecipitates were eluted from the beads with 50 μL of sample loading buffer or FLAG peptides at a concentration of 100 μg/mL. Eluted proteins were separated by SDS-PAGE, and probed by immunoblotting. For detection of proteins, anti-Myc (Sigma), anti-FLAG (Sigma), and anti-V5 (Sigma) antibodies were used.

NMR Spectroscopy

All NMR experiments were recorded at 25 °C using a Bruker AVANCE600 spectrometer, equipped with a 5-mm triple-resonance, z-axis gradient cryoprobe, on samples containing 0.3mM 13C/15N-labeled DCAF1 809–876, 809–902, and 841–883 peptides in 25mM sodium phosphate buffer, pH 6.5, 150 mM sodium chloride, 0.02% sodium azide. Backbone and Cβ resonance assignments of DCAF1 809–876, 809–902 and 841–883 were carried out using 2D 1H-15N HSQC and 3D HNCACB and HN(CO)CACB experiments. Secondary structure elements of these peptides were obtained based on secondary chemical shifts using ΔCα minus ΔCβ with ΔCα and ΔCβ values calculated by subtracting random coil Cα and Cβ shifts from the measured values (47, 48).

Multi-angle Light Scattering

The molecular masses of protein complexes were obtained using an analytical Superdex200 column (1 cm × 30 cm, GE Healthcare) with in-line multi-angle light scattering (HELEOS, Wyatt Technology), variable wavelength UV (Agilent 1100 Series, Agilent Technology), and refractive index (Optilab rEX, Wyatt Technology) detection. Typically, about 100 μL of 2 mg/mL of protein solutions were injected into the column that was pre-equilibrated with a buffer containing 25 mM sodium phosphate, pH 7.5, 150 mM NaCl, 5% glycerol, and 0.02% sodium azide at a flow rate of 0.5 mL/min at room temperature. Light scattering data were analyzed using the ASTRA program (Wyatt Technology).

Circular Dichroism (CD) Spectroscopy

Far-UV (195–250 nm) CD spectra of DCAF1 809–876 WT, L850E/L851E, and L852E/L853E mutants were recorded at a concentration of 3.3 μM in 2.5 mM sodium phosphate buffer, pH 7.5, 15 mM NaCl, at 25°C using a JASCO-810 (Easton, MD) spectrophotometer. Data were collected with 0.5 nm intervals and averaged 10 times. Electron Microscopy (EM) Analysis. The purified CRL4DCAF1 complex composed of RBX1-CUL4A-DDB1-DCAF1 817–1507 at a concentration of 0.8 mg/ml was diluted 50 fold and deposited onto glow-discharged carbon foil grids, blotted with filter paper, and stained with 2% uranyl acetate. The grids were examined at 200 kV with a TF20 electron microscope (FEI, Hillsboro, OR). Images were recorded with a 4K×4K Gatan CCD camera (Gatan, Inc., Warrendale, PA) at a nominal magnification of 50,000x and underfocus values ranging from 1.5 to 3.0 μm.

Image Processing of EM

EM images were processed using the EMAN image analysis software (49). Individual particles were boxed manually with 208 × 208 pixels (2.14 Å/pixel), normalized, and combined to yield one raw image stack file. A total of 1900 individual particle images was selected, band pass-filtered, and aligned with respect to their center of mass. The aligned raw projection images were classified using reference-free multi-variance statistical analysis and averaged within each class.

In Vitro Ubiquitination Assays

Typically, E1 (UBA1, 0.2 μM), E2 (UbcH5b, 2.5 μM), and the E3 ligase (0.2 or 0.4 μM of DDB1-CUL4A-RBX1 complexed with the various DCAF1 constructs as indicated) were incubated at 37 °C with 0.5 μM of NusA-Vpr-ΔC (residue of 1–79), 1 μM of UNG2, and 2.5 μM of His6-FLAG-tagged ubiquitin in a buffer containing 10 mM TrisHCl, pH 7.5, 150 mM NaCl, 5% glycerol, 20 U/mL pyrophosphatase, 2 mM DTT, and 5 mM ATP for various lengths of time. Reactions were stopped by heating the samples in SDS-Laemmli buffer at 95 °C for 5 min. The extent of ubiquitination was determined by immunoblotting with anti-FLAG or anti-UNG2 (Abnova) antibodies, after separation of the reaction mixtures on 4–20% gradient SDS-PAGE, and transfer to nitrocellulose.

Results and Discussion

DCAF1 Proteins Oligomerize when ectopically expressed in insect cells

It is known that several CRLs dimerize via substrate receptor proteins (18, 4042, 50). In addition, the DCAF1 sequence contains a Lissencephaly type 1-like homology motif (LisH) (51) between residues 846 and 876, known to mediate dimerization. It therefore seemed prudent to evaluate whether oligomerization of DCAF1 may occur, especially since during protein expression and purification of a long DCAF1 construct (residues 96–1507) aggregation was observed, with DDB1–DCAF1 complexes eluting in the void volume of a Superdex200 gel-filtration column (Ahn et al., unpublished results). The apparent discrepancy between the expected molecular mass (280 kD) of the complex and the experimental behavior warranted further examination.

In order to assess whether oligomerization of DCAF1 occurred, co-immunoprecipitation of transiently expressed, differentially tagged DCAF1 proteins in insect cells was carried out. Specifically, both N-terminally Myc-tagged DCAF1 and N-terminally FLAG-tagged DCAF1 (residue 96–1507) were transiently co-expressed in SF9 cells, with both constructs containing a V5-tag at their C-termini. Cell lysates were subjected to immunoprecipitation with anti-FLAG antibodies and indeed, interaction between the Myc-tagged and FLAG-tagged DCAF1 proteins was observed (Fig. 1A, lane 1).

Figure 1
DCAF1 oligomerizes via a putative LisH motif IN VIVO

The oligomerization domain was mapped by co-expressing various truncated DCAF1 constructs (residues 572–1507, 817–1507, 876–1396, 987–1396, and 1005–1507), tagged with the V5 epitope at their C-termini together with the FLAG-tagged DCAF1 (residues 96–1507). Immunoprecipitation with anti-FLAG antibodies, and subsequent immunoblotting with anti-V5 antibodies of co-expressed DCAF constructs revealed that the putative LisH motif, comprising residues 846–876, needed to be present for stable interaction (Fig. 1B). In particular, the 876–1396, 987–1396, or 1005–1507 constructs failed to co-immunoprecipitate with the FLAG-tagged DCAF1 96–1507 bait, while 572–1507 or 817–1407 constructs did. This data provided strong evidence for a possible direct interaction between DCAF1 proteins, mediated by residues 817–876 that contain the putative LisH motif. However, association of DCAF1 proteins via other endogenous cellular proteins could not be ruled out. To directly confirm the homo-oligomerization of DCAF1 proteins, the isolated peptide region implicated in the association was investigated as described below.

Secondary Structure of a DCAF1-LisH Peptide in Solution

The secondary structure of the LisH motif containing region of DCAF1 was investigated by NMR. Three different lengths of peptides were prepared, DCAF1 809–876, DCAF1 809–902 and DCAF1 841–883 (Fig. 2). Qualitative structural characterization of the DCAF1 809–876 WT peptide analyzing secondary chemical shifts (ΔδCα–ΔδCβ) clearly suggested that the C-terminal half of the peptide (Phe845–Glu873) exhibited an alpha helical conformation, indicated by a stretch of large positive values of secondary chemical shifts for Glu847–Ser860 and Leu862–Glu873, with a break at Lys861 (Fig. 2A). Most of the residues in the N-terminal half of DCAF1 809–876 also exhibit positive secondary chemical shifts, albeit too small to suggest that a helical structure is present. In order to unambiguously delineate the helix boundaries, the peptide was extended at its C-terminus up to residue 902 (Fig. 2B). The secondary chemical shifts for this longer peptide revealed that the helix terminates at Glu873. Further, a shorter DCAF1 peptide (residues 841–883) also displays similar boundaries for the helical region (Fig. 2C). This data confirms that the putative LisH motif (residues 846–876) is embedded in an alpha helical structure, in agreement with the well-characterized helical LisH motif of LIS1(52, 53). One notable different feature of the region surrounding the LisH motif in DCAF1 is that the residues following the motif display random coil properties. The canonical LisH motif is often followed by an extended coiled-coil segment with a short helical segment connecting these two regions (52, 53). However, these coiled-coil segments do not contribute to dimerization and exhibit random coil properties as isolated fragments (53).

Figure 2
Secondary structure characterization of isolated DCAF1 peptides by NMR

The Region of the Four Leu Repeat is Essential for Alpha-helical Structure and Oligomerization of DCAF1

The LisH motif is present in numerous eukaryotic proteins, frequently N-terminal to WD40 domains (51), and in DCAF1 the prototypical L-X2-L-X3–5-L-X3–5-L sequence (51) is located at position 850–863. Sequence alignment of the pertinent DCAF1 region with orthologous sequences and human LIS1 is provided in Fig. 3A. Interestingly, the LisH motif of DCAF1 contains four Leu residues adjacent to Ile854, the residue that corresponds to the critical Ile15 residue in the dimer interface of the LIS1 structure (52, 54). In order to assess whether these Leu residues are important for oligomer formation, we constructed two mutants of DCAF1, ΔN-L850E/L851E DCAF1-V5 (residues 817–1507 of L850E/L851E DCAF1 with a C-terminal V5-tag; 1LL/EE) and ΔN-L852E/L853E DCAF1-V5 (residues 817–1507 of L852E/L853E DCAF1 with a C-terminal V5-tag; 2LL/EE). Co-expression of either mutant with N-terminally FLAG-tagged and C-terminally V5-tagged DCAF1 (FLAG-DCAF1-V5, residues 96–1507) did not result in efficient co-immunoprecipitation (Fig. 3B, lane 2 and 4). In contrast, WT ΔN-DCAF1-V5 co-precipitated efficiently with FLAG-DCAF1-V5 (Fig. 3B, lane 1). This data suggests that residues within the Leu850–Leu853 stretch are essential for the interaction between different DCAF1 molecules.

Figure 3
The four Leu region is critical for oligomerization of DCAF1

To directly confirm that the four Leu-repeat in the LisH motif of DCAF1 is responsible for oligomerization, both mutations, 1LL/EE and 2LL/EE, were introduced into a Trx-peptide fusion construct and fusion proteins and the DCAF1 809–876 peptide were purified to homogeneity (Fig 3C). The quaternary states of Trx-DCAF1 809–876, Trx-DCAF1 809–876 1LL/EE, and Trx-DCAF1 809–876 2LL/EE were analyzed by a multi-angle light scattering. The experimental molecular mass of Trx-DCAF1 809–876 was derived as 51 kDa, significantly higher than the monomeric theoretical molecular mass (27 kDa) by almost two-fold (Fig. 3D, [triangle]). The estimated molecular masses of the Trx-DCAF1 1LL/EE and 2LL/EE proteins were 33 kDa and 28 kDa, respectively, close to the theoretical monomer molecular masses (Fig. 3D, [diamond] and ● , respectively). For comparison, the unfused Trx protein eluted as a monomer, (experimental and theoretical molecular mass of ~18 kDa, ■). These light scattering data strongly suggest that indeed the LisH motif of DCAF1 is responsible for oligomerization, with the Leu repeat providing critical determinants at the interface. To monitor potential structural changes in the LisH motif upon Leu residue mutation, the Trx-DCAF1 809–876 WT, 1LL/EE, and 2LL/EE fusion proteins were cleaved with TEV protease and DCAF1 peptides purified (Fig. 4A, insert). The circular dichroism (CD) spectrum of the cleaved DCAF1 809–876 WT peptide exhibited two minima at 208 nm and 222 nm, indicating the presence of alpha-helical structure (Fig. 4, circles). The monomeric DCAF1 LL/EE mutants, however, exhibited CD spectra characteristic of random coil structure (Fig. 4, triangles and squares), suggesting that the four Leu repeat is essential for helix formation. For other LisH motif containing genes, it has been reported that disease-related mutations in this motif that abrogate oligomerization, affect protein half-life and alter cellular localization (54, 55). In the present case, WT DCAF1 and LL/EE mutants in transiently transfected HEK293 cells essentially exhibited the same half-life (data not shown).

Figure 4
Secondary structure characterization of WT and mutant LisH motif peptides by CD

The Adaptor-Receptor Complex, DDB1-DCAF1 Dimerizes via the DCAF1 Helical Domain

DDB1 is an adaptor protein that binds to the scaffolding protein complex, CUL4A-RBX1 and the substrate receptor, DCAF1, forming the E3 ubiquitin ligase. To evaluate the quaternary state of the adaptor-receptor complex, DDB1 complexes containing the 817–1507 DCAF1 constructs were purified and analyzed by light scattering (Fig. 5). The observed molecular mass (411 kDa) of this complex is twice its theoretical value, (DDB1, 130 kDa; DCAF1 817–1507, 81 kDa), lending further support to the finding that the region encompassing the LisH motif of DCAF1 mediates dimerization of the DDB1–DCAF1 complexes.

Figure 5
Characterization of the DDB1-DCAF1 complex by SEC-MALS and SDS-PAGE

Electron Microscopy (EM) and 2D Image Analysis Suggests a Dimeric CRLDCAF1 Organization

For electron microscopy, we reconstituted the entire CRL4DCAF1 E3 ubiquitin ligase complex from its four components. As evidenced by SDS-PAGE (Fig. 6A), the four individual proteins in the complex are present in equimolar amounts (CUL4A, 87 kDa; RBX1, 12 kDa; DDB1, 130 kDa; DCAF1 817–1507, 81 kDa) resulting in an overall molecular mass of the complex of 650 kDa by light scattering, confirming its dimeric quaternary state (Fig. 6B). However, some smaller species, possibly sub-complexes, are also observed upon 50 fold dilution to 50 nM concentration (necessary for EM analysis). The larger particles represent the intact complex (Fig. 6C). They are well separated from each other and appear to be homogeneous with similar sizes after uranyl acetate staining. From EM projection images, the estimated diameter of a sphere required for enclosing these large particles is about 180Å, significantly larger than what would be expected for a monomeric, 330kDa CRL4DCAF1 particle. Classification of approximately 1,900 particle images by multivariant statistical analysis from the raw micrographs resulted in the class averages depicted in Fig. 6D. Near-mirror symmetries were observed for a few of the 2D class averages (Fig. 6D), suggesting the presence of a twofold axis in the particles. Therefore, the EM study of CRL4DCAF1 particles agrees well with the biochemical analyses described above.

Figure 6
Electron microscopy of CRL4DCAF1 E3 ligase complexes

Possible Roles of Dimerization of DCAF1 in Ubiquitin Ligase Activity of CRL4DCAF1-Vpr

Dimerization of the CUL4A-RING E3 ligase can impart functional advantages onto the ligase complex, such as permitting productive loading of different size and shape substrates for ubiquitination. Many other ubiquitin E3 ligases dimerize via substrate receptor proteins. For example, in some Skp1-Cdc53/Cul1-F-box (SCF) E3 ligases, a small dimerization domain (D-box) is located within the substrate receptor protein (56, 57). It is believed that dimerization of CRLs imparts variability in the overall geometry of the complexes, permitting ubiquitination of substrates in cis as well as in trans (4044).

The ubiquitination activities of monomeric and dimeric CRL4DCAF1 E3 ligases were tested using DCAF1 817–1507 (dimeric) or DCAF1 1005–1507 (monomeric) constructs (Fig. 7A). Although it is not clear which cellular substrates are targeted by CRL4DCAF1, we previously demonstrated that uracil DNA glycosylase-2 (UNG2) becomes ubiquitinated via CRL4DCAF1 in a Vpr-dependent manner (46). Ubiquitin transfer activity of dimeric CRL4DCAF1 is more than twofold higher than that of a monomeric complex (Fig. 7B. compare lanes 2–4 with 6–8 and lanes 10–12 with 14–16), supporting the notion that dimerization of substrate recognition proteins allows for more efficient ubiquitin transfer. However, other possible reasons for the observed difference could also be related to the missing region between the LisH motif and the start of the WD40 domain in monomeric CRL4DCAF1.

Figure 7
Ubiquitination activity of monomeric and dimeric CRL4DCAF1

Previous structural data of DDB1 have revealed that the relative orientation of CUL4A-binding domain is variable relative to the substrate receptor-binding domains, creating different distances between CUL4A-RBX1 and the substrate. (1921). This also may facilitate ubiquitination of substrates, and dimerization of DCAF1 potentially could add further conformational variability. However, it should be noted that the LisH motif is not commonly present among putative DCAF substrate receptors (58) and further structural and biochemical studies are necessary to fully evaluate the conformational variability in these complexes.

Conclusions

DCAF1, initially identified as a HIV-1 Vpr binding protein (VprBP) and belonging to the large family of WD40 domain containing substrate receptors of CUL4-RING E3 ubiquitin ligases, forms an adaptor-receptor complex with DDB1. Although several of DCAF1’s biological functions have been described, our understanding of its activity in proteasomal-degradation of target substrates still remains refractory. In this report, we demonstrate that a stretch of Leu residues in the LisH sequence motif is essential for oligomerization. The LisH motif is embedded in a region of ca 40 amino acids (~840–880) that possesses helical structure and is located N-terminal to the WD40 domain of DCAF1. This region is important for dimerization of the CRL4 complex and dimeric CRL4DCAF1 appears to possess enhanced Vpr-mediated ubiquitin transfer activity, compared to its mutant monomeric counterpart.

Acknowledgments

This work was supported by the National Institutes of General Medical Sciences (NIH Grant P50GM082251 to A. M. G. and GM085043 to P. Z.).

We thank Dr. Teresa Brosenitsch for critical reading of the manuscript, and Thomas Vu and Mathieu Cuchanski for expert technical assistance. We also thank Dr. Jacek Skowronski for communication of unpublished results.

Abbreviations

Ub
ubiquitin
E1
Ubiquitin-activating enzyme
E2
Ubiquitin-conjugating enzyme
E3
Ubiquitin ligase
ATP
adenosine triphosphate
RING
really interesting new gene
CUL
cullin
CRL
cullin-RING ubiquitin ligase
DDB1
DNA-damage-binding protein 1
DCAF1
DDB1-CUL4A-Associated Factor 1
HIV-1
human immunodeficiency virus-1
Vpr
viral protein, regulatory
LIS1
lissencephaly associated gene 1
LisH
LIS 1 homology
CRL4
DDB1-culling4A-RING ubiquitin ligase
CRL4DCAF1
CRL4 in complex with DCAF1
EM
electron microscopy
SEC-MALS
size-exclusion column chromatography coupled to in-line multi-angle light scattering

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