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. 2024 May 10;63(10):1297–1306. doi: 10.1021/acs.biochem.4c00044

Development of Peptide Displacement Assays to Screen for Antagonists of DDB1 Interactions

Darren Yong †, Shabbir Ahmad †, Mark F Mabanglo ‡, Levon Halabelian †,§, Matthieu Schapira †,§, Suzanne Ackloo †, Sumera Perveen †, Pegah Ghiabi †, Masoud Vedadi ‡,§,*
PMCID: PMC11112733  PMID: 38729622

Abstract

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The DNA damage binding protein 1 (DDB1) is an essential component of protein complexes involved in DNA damage repair and the ubiquitin-proteasome system (UPS) for protein degradation. As an adaptor protein specific to Cullin-RING E3 ligases, DDB1 binds different receptors that poise protein substrates for ubiquitination and subsequent degradation by the 26S proteasome. Examples of DDB1-binding protein receptors are Cereblon (CRBN) and the WD-repeat containing DDB1- and CUL4-associated factors (DCAFs). Cognate substrates of CRBN and DCAFs are involved in cancer-related cellular processes or are mimicked by viruses to reprogram E3 ligases for the ubiquitination of antiviral host factors. Thus, disrupting interactions of DDB1 with receptor proteins might be an effective strategy for anticancer and antiviral drug discovery. Here, we developed fluorescence polarization (FP)-based peptide displacement assays that utilize full-length DDB1 and fluorescein isothiocyanate (FITC)-labeled peptide probes derived from the specific binding motifs of DDB1 interactors. A general FP-based assay condition applicable to diverse peptide probes was determined and optimized. Mutagenesis and biophysical analyses were then employed to identify the most suitable peptide probe. The FITC-DCAF15 L49A peptide binds DDB1 with a dissociation constant of 68 nM and can be displaced competitively by unlabeled peptides at sub-μM to low nM concentrations. These peptide displacement assays can be used to screen small molecule libraries to identify novel modulators that could specifically antagonize DDB1 interactions toward development of antiviral and cancer therapeutics.

Introduction

DDB1 is a multidomain protein involved in DNA repair and protein ubiquitination as part of UV-DDB and E3 ubiquitin ligase complexes, respectively. The UV-damaged DNA-binding protein (UV-DDB) is a heterodimeric protein consisting of DDB1 and DDB2 subunits. This complex recognizes DNA lesions induced by UV damage during global genome nucleotide excision repair1 and recruiting proteins to initiate DNA repair.2−7 E3 ubiquitin ligases, along with ubiquitin-activating (E1) and ubiquitin-conjugating (E2) enzymes, function in the stepwise ubiquitination of substrate proteins destined for degradation by the 26S proteasome (Figure 1A).8,9 Ubiquitination is conserved in eukaryotes and is central to many fundamental cellular processes such as transcription regulation, protein sorting and localization, cell cycle control, and other signaling pathways.10

Figure 1.

Figure 1

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Ubiquitination and the DDB1–DCAF interaction in CRL4 E3 ligase. (A) The process of protein substrate ubiquitination is carried out by ATP-dependent E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin ligases. The CRL4 E3 ligase uses Rbx/ROC (purple oval) to bind ubiquitin-charged E2 (light blue square), allowing the transfer of ubiquitin to substrate proteins (orange diamond). The protein substrate is poised for ubiquitination by the CRL4 E3 ligase using DDB1 (green triangle) as a scaffolding adaptor and DCAFs (dark green rectangle) as substrate receptors. (B) Structural model of the CRL4 E3 ligase composed of CUL4 (gray), Rbx1 (purple), DDB1 (green), DCAF15 (dark teal), DDA1 (red), and the protein substrate RBM39 (orange). The model was generated by using PDB 4A0C (Rbx1), 4A0K (Cullin 4A, DDB1), and 6Q0R (DCAF15, DDA1, RBM39). (C) The canonical binding pocket at the interface between the BPA and BPC domains of DDB1 (green). The synthetic peptides based on the H–L–H (like) motifs of DDB1 interacting partners are superimposed within the canonical binding site (DCAF1, dark teal; DCAF15, orange; DDB2, dark blue; DCAF1: magenta; HBx, light green). DDA1 is shown in red cartoon, but with the DDA1 peptide used in this study highlighted in wheat. As in B, DDA1 binds a long and shallow surface on one side of DDB1’s BPA domain. (D) H–L–H (like) motifs of CRBN and CDK12 adopt different binding modes in the canonical binding site. The CRBN (blue) and CDK12 (pink) peptides are shown in the binding pocket. Shown in the box are the same peptides, rotated by −20° along the y-axis of the original image. (E) Structure-based alignment of the 13-residue, α-helical, canonical site binding motifs of DDB1 interactors, including those of viral proteins HBx and SV5 V. The PDB ID of each represented protein is indicated on the left labels. Residues are color-coded as follows: green, polar; yellow, hydrophobic; blue, basic; red, acidic; purple, Gly or Pro. The bar graph represents the frequency of the indicated residues within the group of analyzed proteins. (F) The DDB1 canonical binding pocket with bound H–L–H (like) peptide motifs of the 15 interactors listed in E, superimposed: HBx, teal; DCAF12, dark teal; DCAF6, magenta; DCAF9, yellow; WHx, pink; DCAF5, gray; DCAF8, purple; DCAF4, orange; DCAF15, wheat; DCAF1, deep olive; CSA, light green; DDB2, beige; SV5 V, light purple.

Out of about 600 E3 ubiquitin ligases in humans, the RING-finger E3 ubiquitin ligases comprise the largest family. Within this family, a subset called Cullin-RING ligases (CRLs) utilizes DDB1 as a specific adaptor component of the complex that binds to substrate receptors (Figure 1A,B).9 Among the many cognate substrate receptors of DDB1 are Cereblon (CRBN) and DDB1 and CUL4-associated factors (DCAFs).11−16 CRBN is a component of CUL4-DDB1-CRBN (CRL4CBN) E3 ligase that is widely used in the development of proteolysis targeting chimeras (PROTACs).17 Thalidomide-based immunomodulatory drugs (IMiDs) exert their anticancer effects on myeloma and other cancer cells through CRBN. Each IMiD binds to CRBN and alters the substrate specificity of CRL4CBN, resulting in the breakdown of Ikaros and Aiolos. These transcription factors regulate the development of hematopoietic and immune cells, including CD4+ T cells.18,19

The DDB1-CUL4 complex is predicted to assemble with a significant number of putative DCAFs to recognize a wide array of substrate proteins.20−22 The specific function of many DCAFs remains poorly understood, and it is unclear how each DCAF is selectively recognized by DDB1.15 One of the most studied DCAFs, DCAF1 associates with CUL4A and DDB1 to form the CRL4DCAF1 complex. Human immunodeficiency viruses 1 and 2 can hijack CRL4DCAF1 using viral proteins Vpr and Vpx, respectively, which bind DCAF1 like a decoy to capture host antiviral factors and other proteins, including SAMHD1, SIRT7, Dicer, and UNG2.23−28 The crystal structure of DDB1-DCAF1-Vpr-UNG2 revealed how the viral protein Vpr mimics DNA binding to UNG2, providing the structural basis for viral reprogramming of CRL4DCAF1 to modify the host cell environment and promote viral replication and pathogenesis. Viral mimicry of endogenous DCAF1 substrates has been shown to induce cell cycle arrest.27,28

The structure of DDB1 consists of three seven-bladed β-propeller domains (BPA, BPB, and BPC) and a helical C-terminal domain (CTD) (Figure 1C). The BP domains cluster together to form a star-shaped structure with a large interactor binding pocket between the BPA and BPC domains (canonical site).16 DDB1 associates with the N-terminal portion of CUL4 using the BPB domain that is connected to the BPC domain by flexible loops (Figure 1B,C). DCAFs and other DDB1-interacting receptors bind the canonical site through a helix–loop–helix (H–L–H) or a similar motif (Figure 1C).30 DDB1 also binds CRL4 stabilizing elements such as DET1 and DDB1-associated protein 1 (DDA1). DDA1 is a chromatin-associated scaffold protein that regulates CUL4-based E3 ligases during DNA replication and repair.31 However, unlike other interactors, DDA1 binds DDB1 through a long and shallow binding surface on BPA, part of which is located near the intersection of the three BP domains (noncanonical site) (Figure 1C). As a stabilizing factor, DDA1 simultaneously maintains interactions with other substrate receptors to enhance the integrity of the complex.32

The canonical binding site of DDB1 is large and can accommodate at least three distinct binding modes of H–L–H (like) motifs. Within this site, most interactors engage BPC using a 13-residue α-helix (Figure 1C). Other interactors such as CRBN and CDK12 bind the same general area on BPC but engage different residues or interact with BPB using extended peptide regions (Figure 1C,D). Structural alignment of these 13-residue α-helices reveals a weak sequence identity (Figure 1E). In general, positions 1 and 10 of the helix tend to have large polar residues, such as Asn and Gln, respectively. Positions 2, 3, 5, 6, and 11 are primarily occupied by hydrophobic residues, with Leu being the most common. Positions 7, 8, and 9 are primarily basic and often include Arg to complement the charge of the acidic DDB1 pocket. This weak motif signature is exploited by viral proteins such as Simian Virus 5 V (SV5 V) and Hepatitis B virus X (HBx) (Figure 1E,F).15,29 Like Vpr, SV5 V and HBx closely mimic the binding modes of DCAF peptides (RMSD of 0.6–0.8 Å) (Figure 1F).33

Given its involvement in diverse cellular processes, DDB1 has become an attractive target for the development of therapeutic agents against cancer and viral diseases.34−36 Here, we employed biophysical methods to characterize the binding of DCAF15 (33–54), DDB2 (101–126), DCAF1 (1046–1069), CRBN (219–249), and DDA123−37 or CDK12 (1046–1069) to DDB1. We identified a minimal peptide sequence that can be fluorescently labeled and used with DDB1 in optimized fluorescence polarization (FP)-based assays with potential application in the screening of small molecule libraries to identify antagonists of DDB1 interactions.

Materials and Methods

Peptides and Reagents

The modified and unmodified peptides (Table S1) used in this study were commercially synthesized by GenScript. The purity (>98%) of the peptides was assessed by high-performance liquid chromatography (HPLC). The peptide probes were modified either N- or C-terminally with fluorescein isothiocyanate (FITC). The concentration of the peptides was determined based on the molecular weights. All other chemicals, e.g., buffer components, additives, detergents, and dimethyl sulfoxide (DMSO) were purchased from common commercial sources.

Protein Expression and Purification

DNA fragment encoding full-length DDB1 (residues M1-H1140, isoform 1) was amplified by PCR and subcloned into pFBOH-MHL expression vector. The resulting plasmid was transformed into DH10Bac-competent Esherichia coli (Invitrogen), and a recombinant viral DNA bacmid was purified and followed by a recombinant baculovirus generation in sf9 insect cells. The sf9 cells grown in HyQ SFX insect serum-free medium (Thermo Scientific) were infected with 10 mL of P3 viral stock per 0.8 L of suspension cell culture and incubated at 27 °C using a platform shaker set at 100 rpm. The cells were collected 72 h postinfection, when viability dropped to 70–80%. Harvested cells were resuspended in binding buffer containing 20 mM Tris-HCl, pH 7.5 containing 500 mM NaCl, 5 mM imidazole and 5% glycerol, 10 mM β-mercaptoethanol, and 1× protease inhibitor cocktail (100× protease inhibitor stock in 70% ethanol (0.25 mg/mL aprotinin, 0.25 mg/mL leupeptin, 0.25 mg/mL pepstatin A, and 0.25 mg/mL E-64) or Roche complete EDTA-free protease inhibitor cocktail tablet). The cells were lysed chemically by rotating for 30 min with NP40 (final concentration of 0.6%) and 22.5 U/mL benzonase nuclease (in-house), followed by sonication at a frequency of 7.5 (10” on/7” off) for 6 min (Sonicator 3000, Misoni). The crude extract was clarified by high-speed centrifugation (60 min at 28 000 g at 4 °C). The clarified lysate containing the his-tagged protein was then loaded onto an open column containing pre-equilibrated Ni-NTA (Qiagen) for affinity purification for 3 h. The column was then washed with binding buffer containing 30 mM imidazole after washing with 1 mM D-Biotin in PBS. The DDB1 protein was then eluted with a binding buffer containing 250 mM imidazole. The eluted protein was further purified by size exclusion chromatography on a HiLoad Superdex200 26/600 column, pre-equilibrated with 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5% glycerol, and 10 mM β-mercaptoethanol, using an ÄKTA Pure System (Cytiva). The purity of the fractions was assessed on SDS-PAGE gels and pure fractions were pooled, concentrated, and flash frozen.

FP Assay Development

All FP assays were performed as previously reported37 with some modifications. Assays were performed in a final volume of 20 μL in black flat bottom 384-well polypropylene microplates (Greiner ref. no. 784209). Protein and tracer peptide mixtures were incubated at room temperature for 30 min before the FP signal was measured on a Synergy H1 (BioTek) plate reader using 485/20 nm excitation and 528/20 nm emission filters.

The buffer conditions for each DDB1-peptide pair in the FP-based assay were individually determined for the optimal signal-to-noise ratio within physiological parameters. Saturation curves were determined using 40 nM of FITC-labeled peptides derived from DCAF15 (33–54), DDB2 (101–126), DCAF1 (1046–1069), CRBN (219–249), and DDA123−37 or CDK12 (1046–1069) and titrating up to a final concentration of 10 μM of DDB1. The FP values were plotted against protein concentration, and the dissociation constants (Kd values) were obtained by analyzing the data using GraphPad Prism as previously described.37 The competitive peptide displacement assays were performed using the corresponding unlabeled peptide and/or the orthogonal unlabeled peptide to compete out the respective tracer peptide using a protein concentration below saturation (40–80% saturation, depending on the peptide Kd). The resulting dose–response curve was fitted to a four-parameter sigmoidal model by using GraphPad Prism to obtain the K displacement (Kdisp) values.

The optimized FP assay performance under a high-throughput format was validated by determining the Z′ factor as previously described.38

All experiments were performed in triplicate (n = 3), and plotted values represent the average of three replicates ± standard deviation. Data were plotted using GraphPad Prism 8.4.3 (GraphPad, La Jolla, CA).

Investigating the H–L–H Consensus Sequence for DDB1 Interaction

The coordinates of 15 DDB1-containing complexes were retrieved from the PDB and superimposed in PyMol to create a structural alignment of the H–L–H (like) peptides. Residues that were structurally aligned in the BPA-BPC binding pocket were then analyzed to define a peptide consensus sequence.

Results and Discussion

Identification and Optimization of FP Assay Condition

As a first step in the development of a screening method for small molecule compounds targeting DDB1 interactions, we prepared six different H–L–H (like) motifs of cognate DDB1 interactors for use in FP-based peptide displacement assays (Figure 1F). FITC-labeled and unlabeled peptides were commercially synthesized based on the sequences of five DDB1 interacting proteins: DCAF15, DCAF1, DDB2, CRBN, and CDK12. A pair of FITC-labeled and unlabeled peptides was also synthesized based on the sequence of the noncanonical site-binding region of DDA1. In the FP experiment, complexation of DDB1 with FITC-labeled peptide results in increased fluorescence polarization due to slower tumbling of the bound peptide relative to the free labeled peptide.

To determine the optimal assay condition for each peptide, various buffers and additives were explored using a near physiological pH range of 6.0 to 8.0 (Figures 2A–I and S1–S5). Good FP signal-to-noise were observed in HEPES buffer for most peptides. For DCAF15 and DDB2 peptides, bis-tris propane (BTP) was also a suitable buffer for assay optimization (Figures 2 and S2). The concentration of HEPES or BTP was then optimized for each peptide. Next, the effects of other buffer components such as salt, detergent, reducing agent, and DMSO were tested (Figures 2 and S1–S5). For the DDB1–DCAF15 interaction, no significant dependence on salt concentration was observed up to 100 mM (Figure 2D). In contrast, the DDA1 and DCAF1 peptide interaction with DDB1 were abolished at high salt concentration (>10 mM NaCl) (Figures S1D,E, and S3C). The use of detergents and additives significantly improves the signal-to-noise ratio and stability of FP signal by limiting the nonspecific binding and aggregation of the tracer peptide and compounds in high throughput screening.39 Several detergents and reducing agents were assessed at a wide concentration range for the selected peptides (Figures 2E–H, S1G–I, S2G,H, and S3E–H). No significant effect on the FP signal was observed for Triton X-100 at 0.01% and was selected for all assays. Albeit varied among assays, DTT at low concentrations (2–5 mM) also had little effect on the signal readout. All of the assay conditions are summarized in Table 1. Under the optimal FP assay conditions for each peptide, we measured their dissociation constants (Kd) by titrating the peptide with increasing concentrations of DDB1. Besides CDK12 peptide, which did not show binding, all other tested peptides bound to DDB1 with low nanomolar affinities under specified assay conditions (Table 1 and Figure S6A–E). DDA1 and DCAF1 peptides did not bind well to DDB1 in the presence of NaCl, even at low concentrations; thus, their final assay buffers did not include salt (Table 1 and Figures S1D,E, and S3C). Notably, CRBN and DCAF15 peptides bound to DDB1 with high affinity (Kd of 5 and 13 nM, respectively) (Table 1 and Figure S6A–E).

Figure 2.

Figure 2

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Optimization of FP-based peptide displacement assay conditions for measuring the interaction between DDB1 and the FITC-labeled DCAF15 (33–64) peptide. (A) Buffer, (B) pH, (C) buffer concentration, (D) salt concentration, detergents (E) Triton X-100 and (F) Tween 20, reducing agents (G) DTT and (H) TCEP, and (I) DMSO concentration.

Table 1. Binding Parameters and Individual Assay Conditions for the Interactions between Each FITC-Labeled Peptide and DDB1a.

peptide binding site Kd (nM) Kdisp (nM) buffer [NaCl] (mM) pH
DDA1-FITC (24–39) Noncanonical 27 ± 3 129 ± 8 10 mM HEPES, 0.01% Triton X-100, 2 mM DTT - 7.0
DCAF15-FITC (33–64) Canonical 13 ± 0.5 83 ± 2 40 mM BTP, 0.01% Triton X-100, 5 mM DTT 150 7.5
DDB2-FITC (101–126) Canonical 79 ± 14 1649 ± 90 40 mM BTP, 0.01% Triton X-100, 5 mM DTT 150 7.5
FITC-CRBN(219–249) Canonical 5 ± 0.6 115 ± 6 40 mM BTP, 0.01% Triton X-100, 5 mM DTT 100 7.0
FITC-DCAF1 (1046–1069) Canonical 109 ± 16 1521 ± 90 40 mM HEPES, 0.01% Triton X-100, 2 mM DTT - 7.5
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a

Errors are represented as ±SE. Data for CDK12 were not included because no significant binding with DDB1 was observed. Data are from Figure 2 and Figures S1–S4.

In crystal structures, the H–L–H (like) motifs of CRBN and DCAF15 adopt distinct binding configurations, which indicates that the canonical site is large enough to accommodate them (Figure 1D,F).26 The DDB2 peptide contains a full H–L–H motif bound with similar affinity (Kd of 79 nM) to CRBN and DCAF15 peptides. On the other hand, the DCAF1 peptide, which contains only the first 13-residue α-helix and the loop segment of the H–L–H motif, showed lower affinity (109 nM) (Table 1), indicating that full H–L–H motifs maximize contact with the canonical pocket of DDB1. Lastly, the CDK12 peptide failed to bind under any of the conditions tested (Figure S5A,B). This was not surprising as it has been reported that CR8 binding to the CDK12 active site facilitates interaction of CDK12–DDB1 and the CDK12 peptide may not represent the interaction interface (Figure 1D).30

Competitive Peptide Displacement Assays Using Unlabeled Peptides

The FP saturation binding experiments above provided useful information about binding affinity, signal range, and optimum protein concentrations to set up competitive peptide displacement assays using unlabeled peptides. Addition of an unlabeled peptide dissociates the DDB1-(FITC)-peptide complex, resulting in decreased fluorescence polarization. Competition assays were conducted using the optimized condition for each DDB1-peptide pair (Table 1) and under a general condition (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.01% Triton X-100, and 1 mM DTT) to enable cross comparison. This general condition provided good signal-to-noise ratios and reproducibility for most DDB1-peptide pairs (Figure 3A–C) with Kdisp values similar to those in Table 1 except for DDB2. However, the presence of salt (150 mM NaCl) in this condition abolished DDA1 and DCAF1 peptide interactions with DDB1 (Figure 3A–C and Table 1). For the DDB2 peptide, binding affinity improved fivefold (16 nM), while the dynamic range remained the same under the general conditions (Table 1 and Figures 3C and S6C).

Figure 3.

Figure 3

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Binding and displacement parameters for DDB1 interacting peptides tested under the experimentally established general condition containing 20 mM HEPES, pH 7.5, 150 mM NaCl, 0.01% Triton X-100, and 1 mM DTT. Errors are represented as ±SE. Binding saturation curves (A–C) and competitive peptide displacement by the corresponding unlabeled peptides (D–F) for DCAF15-FITC (A,D), FITC-CRBN (B,E), and DDB2-FITC (C,F).

To achieve a suitable dynamic range in the competitive displacement assay, the DDB1 concentration was chosen well above the Kd to attain a binding saturation of more than 50% for the FITC-labeled peptides. Under both individual and general conditions, unlabeled peptides displaced the corresponding labeled peptides (Figures 3D–F and S7A–E). Based on derived values of Kdisp under both conditions (Table 1 and Figures 3D–F and S7A–E), it appears that the displacement of labeled peptides is optimal under the general condition.

From Kdisp values, we calculated the Ki values for the respective unlabeled peptides.41 Similar affinities were observed for unlabeled DCAF15 and CRBN peptides under the general conditions as compared to those of corresponding labeled peptides (Table 2). The Ki for unlabeled DCAF15 (9.0 nM) and CRBN (1.6 nM) peptides are in the same order as the Kd measured for FITC-labeled DCAF15 (8.0 nM) and CRBN (4.0 nM) peptides (Table 2 and Figure 3A,B). In contrast, the calculated Ki (95 nM) shows that unlabeled DDB2 peptide displays lower affinity compared to that of FITC-labeled DDB2 (Kd of 16 nM). To assess whether the presence of FITC label could have contributed to DDB1 affinity for these FITC-labeled peptides nonspecifically, we used short random peptide sequences with FITC labels at the N- or C-terminus (FP1: FITC-AHA (6-aminohexanoic acid linker) -GGERGLV and FP2: YPKRIAK-AHA-FITC) and measured the FP signal. Incubation with DDB1 did not result in increased fluorescence polarization for any of these random FITC-labeled peptides (Figure S8A), indicating that FITC did not cause interaction with DDB1, and that DDB1 did not bind the random peptides nonspecifically. It is unclear why FITC-labeled DDB2 peptide has higher affinity to DDB1 than its unlabeled counterpart. Conversely, we also tested the binding of DDB1-interacting FITC-labeled peptides to PRMT3, an unrelated methyltransferase, as a negative control to verify any potentially nonspecific binding of interacting peptides. DCAF15 (33–54) and CDK12 peptides exhibited a low level of interaction with PRMT3, while DDA1, DCAF1, CRBN, and DDB2 showed no significant affinity (Figure S8B).

Table 2. Peptide Displacement Assays for CRBN, DDB2, DCAF15 (33–64), and the Lower Affinity DCAF15 (33–54) L49A Mutanta.

labeled peptide unlabeled peptide Kdlabeled Kdisp Kiunlabeled Kdisp60% Ki60% Kdisp80% Ki80%
FITC-CRBN (219–249) CRBN (219–249) 4±0.4 22±2 1.6±0.03 - - - -
DDB2 (101–126)-FITC DDB2 (101–126) 16±3.6 867±90 95±0.4 - - - -
DCAF15-FITC (33–64) DCAF15 (33–64) 8±0.3 58±6 9±0.01 - - - -
DCAF15 (33–54) L49A CRBN (219–249) - - - 176±15 43±3 310±26 28±4
DDB2 (101–126) - - - 415±67 136±0.5 788±73 130±3
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a

Displacement assays of FITC-labeled DCAF15 (L49A) using orthogonal, unlabeled CRBN and DDB2 peptides were also performed. The peptide displacement assays were performed under general conditions (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.01% Triton X-100, and 1 mM DTT). The Ki values for the respective unlabeled peptides were calculated from Kdisp using a method described earlier.41

Identification of a Suitable Peptide Probe for DDB1

FITC-labeled peptides were tested for interaction with DDB1 under the general conditions (Figure 3A–C and Table 2). The CRBN, DCAF15, and DDB2 peptides showed high affinity to DDB1 with Kd values in the low nM range (Figure 3A–C), while DCAF1 and DDA1 peptides did not bind to DDB1 (data not shown). Competitive peptide displacement experiments were performed for labeled CRBN, DCAF15, and DDB2 peptides with the corresponding unlabeled peptides (Figure 3D–F). Of these, CRBN and DCAF15 labeled peptides showed high affinity toward DDB1 with a reasonable signal-to-noise ratio.

Due to the high affinity interaction between DDB1 and some DCAF peptides, disrupting the complex can be difficult. Peptide probes with various affinities are ideal in FP assays for better rank-ordering of small molecules and screening. On the other hand, peptide probes of weak affinity require a high protein concentration to achieve a reasonable binding saturation in FP assays. Since the DDB1-interacting peptides are completely encompassed by the BPA-BPC clam shell (Figure 1C,E), we introduced mutations on the DCAF15 and DDB2 peptides that would decrease the buried surface area upon complexation with DDB1, thereby weakening affinity. To select the desirable mutations, we first assessed per residue binding contributions from the peptide to the DDB1 binding pocket. From available structures, the buried surface area (BSA), electrostatic forces, and intra/intermolecular bonds were calculated using the PDBePISA web server. Evolutionary conservation was calculated from a sequence alignment of full-length proteins using MUSCLE from representative organisms ranging from vertebrates and invertebrates. In conclusion, L49 in DCAF15 and L106 and/or H119 in DDB2 were predicted to contribute the most to binding and, therefore, were chosen for mutagenesis.

A relatively mild L49A mutation was introduced to the DCAF15 peptide. This mutation decreased the affinity eightfold (Kd of 68 nM, Figure 4A,B). For the DDB2 peptide, two single mutations (L106A and H119A) were introduced. The H119A mutation produced a mild effect, increasing Kd by only 1.5-fold (21 nM, Figure S9A). Although the FITC-labeled DDB2 H119A peptide displayed good affinity toward DDB1, the unlabeled counterpart was less effective in displacing the labeled peptide, as was also observed with both labeled and unlabeled wild-type DDB2 peptides (Figures 3F and S9B). On the other hand, the DDB2 L106A mutant had 10-fold lower affinity than wild-type DDB2 peptide, requiring more protein to obtain a satisfactory dynamic range (Figures 3C and S9A,C). Thus, the DCAF15 L49A mutant but not the DDB2 mutants was characterized further for use in FP assays.

Figure 4.

Figure 4

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Effect of the L49A mutation of FITC-labeled DCAF15 (33–64) on peptide binding to DDB1. (A,B) The L49A mutation reduced the binding affinity and modulated binding saturation. The Kdisp values derived for the (C,D) unlabeled DCAF15 (33–64) WT peptide at various saturation levels were not significantly dependent on the binding saturation, whereas the (E,F) strong correlation was evident for the lower affinity DCAF15 (33–54) L49A mutant peptide. While the Y-axis unit for A and B are ΔmP, the percent of maximum values are indicated on each plot as well.

The correlation between binding saturation and Kdisp was analyzed for both the high affinity DCAF15 peptide and the corresponding mutant of lower affinity, DCAF15 L49A. Peptide displacement assays were performed at different binding saturation levels for each peptide probe (Figure 4A,B). As expected, the Kdisp values for the DCAF15 peptide showed little dependence on the DDB1 saturation level, whereas more pronounced variations were observed for DCAF15 L49A at various DDB1 saturation levels (Figure 4C–F). To further demonstrate the utility of FITC-labeled DCAF15 L49A peptide as a probe, we used the orthogonal CRBN and DDB2 unlabeled peptides in competitive displacement assays. Both unlabeled peptides displaced FITC-labeled DCAF15 L49A peptide with respective Kdisp values of 176 and 415 nM at a 60% binding saturation level (Figure 5A, B and Table 2).

Figure 5.

Figure 5

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Competitive displacement of the FITC-labeled DCAF15 (33–54) L49A peptide by unlabeled peptides. (A) CRBN and (B) DDB2 peptides. The assays were performed under 60% and 80% binding saturations.

Finally, we tested the stability of the FP signal with DCAF15 L49A and DDB1 in the presence of increasing amounts of DMSO. Concentrations of >2% DMSO significantly reduced the signal (Figure 6A). In addition, monitoring the signal over time showed no significant change over 2 h of incubation (Figure 6B). Thus, the FP assays may be performed in the presence of 2% DMSO over 30 min. A Z-factor of 0.73 (Figure 6C) also indicates that the assay is suitable and can be efficiently employed in a high-throughput format for screening small molecules that disrupt DDB1–DCAF interactions.

Figure 6.

Figure 6

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FP-based peptide displacement assay optimization and validation. (A) Effect of DMSO concentration. Decrease in FP signal was observed at higher than 2% DMSO. (B) Time course of the FP assay monitored for 2 h showed signal stability. (C) Assay reproducibility. Z-factor (0.73) was determined for the FP-based peptide displacement assay using DCAF15 (33–54) L49A peptide at 60% saturation under general condition of 20 mM HEPES, pH 7.5, 150 mM NaCl, 0.01% Triton X-100, 1 mM DTT, and 2% DMSO.

The displacement assay using DCAF15 L49A peptide has a suitable dynamic range to identify DDB1 binding antagonists, potentially with Kd values of low μM to low nM. In general, the Kd values of the F-labeled peptides and the concentration of DDB1 in the assay (% saturation of DDB1) could affect the dynamic range. In all experiments the FP-labeled peptides were titrated by increasing concentration of DDB1, and a concentration with maximum signal was considered saturation (100%). To allow better competition of unlabeled peptides (a surrogate for DDB1-binding small molecules) with FP-labeled peptide, we performed the displacement assays at protein concentrations as low as 40% of the saturation of DDB1 with an acceptable signal-to-noise ratio (Figure 4E).

In summary, using F-DCAF15 L49A peptide which showed lower affinity (Kd of 68 nM) than wild-type DCAF15 (33–64) (Kd of 8 nM) (Figure 4A,B) allowed better competition of unlabeled peptides (Table 2) and potentially future screening of small molecules. In addition, performing the displacement assays at 40% or 60% saturation of DDB1 (Figure 4E,F) allowed better competition and a wider range of detection, improving the dynamic range. The Kdisp values determined at 80% saturation of proteins are 5–20 times higher than the Kd values for the peptides (Tables 1 and 2). However, using lower DDB1 saturation conditions resulted in lower Kdisp values (closer to the Kd value of the F-labeled peptide), which indicates better competition conditions. Under typical screening concentration of compounds (e.g., 50 μM), the assay should be potentially useful in detecting low nanomolar to possibly low micromolar small molecules.

Conclusion

DDB1 is an essential component of a subset of Cullin-RING E3 ligases, including CRL4CRBN and CRL4DCAF1. Due to their wide substrate scope and the ability of certain viruses to hijack the CRL4DCAF1 complex to target antiviral host proteins for degradation, targeting DDB1 interactions with protein receptors has broad implications in drug discovery. In this work, we developed and optimized an FP-based assay that utilizes full-length DDB1 and a fluorescently labeled peptide fragment of its interactor, DCAF15. The chosen DCAF15 peptide probe was deduced from the biophysical characterization of H–L–H (like) motifs of known DDB1 interactors that directly engage its canonical and noncanonical binding pockets. We demonstrated via mutagenesis and competitive peptide displacement assays that the DDB1-(FITC)-DCAF15 L49A peptide pair is of suitable affinity to achieve binding saturation at reasonable DDB1 concentrations, suitable for high-throughput screening, and development of modulators of DDB1 activity.

Acknowledgments

Funding for the Ontario Institute for Cancer Research is provided by the Government of Ontario. Masoud Vedadi was supported by OICR Senior Investigator award. The Structural Genomics Consortium is a registered charity (no: 1097737) that receives funds from Bayer AG, Boehringer Ingelheim, Bristol Myers Squibb, Genentech, Genome Canada through Ontario Genomics Institute [OGI-196], EU/EFPIA/OICR/McGill/KTH/Diamond Innovative Medicines Initiative 2 Joint Undertaking [EUbOPEN grant 875510], Janssen, Merck KGaA (aka EMD in Canada and US), Pfizer, and Takeda. D.Y. and P.G. acknowledge CQDM Quantum Leap award.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.biochem.4c00044.

  • Modified and unmodified peptide sequence table; buffer components and additive optimization figures for DDA1, DDB2, DCAF1, and CRBN peptides; interaction analysis by FP assay between CDK12 peptide and DDB1; FP-based binding assays for the FITC-labeled peptides to DDB1; FP-based peptide displacement assays by the corresponding unlabeled peptides; nonspecific binding of FITC-labeled peptides; FP-based binding and peptide displacement assay for the DDB2 mutants (PDF)

Author Contributions

∇ D.Y. and S.A. contributed equally. D.Y. contributed to investigation, methodology, visualization, data curation, writing of original draft, and review and editing; S.A. contributed to investigation, visualization, writing of original draft, and review and editing; M.F.M. contributed to visualization, writing of original draft, and review and editing; L.H. contributed to resources and review and editing; M.S. contributed to resources and review and editing; S.A. contributed to resources and review and editing; S.P. contributed to investigation and review and editing; P.G. contributed to investigation and review and editing; M.V. contributed to conceptualization, supervision, writing of original draft, and review and editing.

The authors declare no competing financial interest.

Supplementary Material

bi4c00044_si_001.pdf (547.8KB, pdf)

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Supplementary Materials

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