Structure of the Brd4 ET domain bound to a C-terminal motif from γ-retroviral integrases reveals a conserved mechanism of interaction

Abstract

The bromodomain and extraterminal domain (BET) protein family are promising therapeutic targets for a range of diseases linked to transcriptional activation, cancer, viral latency, and viral integration. Tandem bromodomains selectively tether BET proteins to chromatin by engaging cognate acetylated histone marks, and the extraterminal (ET) domain is the focal point for recruiting a range of cellular and viral proteins. BET proteins guide γ-retroviral integration to transcription start sites and enhancers through bimodal interaction with chromatin and the γ-retroviral integrase (IN). We report the NMR-derived solution structure of the Brd4 ET domain bound to a conserved peptide sequence from the C terminus of murine leukemia virus (MLV) IN. The complex reveals a protein-protein interaction governed by the binding-coupled folding of disordered regions in both interacting partners to form a well-structured intermolecular three-stranded β sheet. In addition, we show that a peptide comprising the ET binding motif (EBM) of MLV IN can disrupt the cognate interaction of Brd4 with NSD3, and that substitutions of Brd4 ET residues essential for binding MLV IN also impair interaction of Brd4 with a number of cellular partners involved in transcriptional regulation and chromatin remodeling. This suggests that γ-retroviruses have evolved the EBM to mimic a cognate interaction motif to achieve effective integration in host chromatin. Collectively, our findings identify key structural features of the ET domain of Brd4 that allow for interactions with both cellular and viral proteins.

Keywords: BET family transcription factor; ET binding motif (EBM); NMR; protein–protein interaction; retroviral integration.

Fig. 1.

Domain maps of Brd4 (A) and MLV integrase (B); the ET-binding motif (EBM) is at the extreme C terminus (yellow). (C) Weblogo (weblogo.berkeley.edu/) of sequence conservation at the C terminus of γ-retroviral integrases, with the EBM peptide used in these studies indicated below; residues 390–405 are highly conserved. β strands formed upon binding the ET domain are indicated by block arrows (see text).

Fig. S1.

Sequence conservation of the ET domain and potential EBM sequences. (A) Sequence conservation of the ET domains from human Brd4 (NM_058243), human Brd3 (NM_007371), human Brd2 (NM_005104), mouse Brd4 (NM_198094), rat Brd4 (NM_001100903), clawed frog Brd4 (NM_001091821), zebrafish Brd4 (NM_001111281), and fruit fly Brd4 (NM_167144). Dagger and asterisk symbols below alignment indicate residues mutated to disrupt the hydrophobic and electrostatic interactions, respectively. (B and C) Lowest-energy conformer from the solution structure of the complex between Brd4 ET and MLV IN EBM showing the electrostatic potential map of the EBM as a transparent molecular surface with sticks highlighting residues involved in the interaction. ET domain residues mutated to disrupt the interaction with MLV IN and host factors are shown in red. (B) Black dotted lines indicate likely ion pair interactions between positive and negative side chains at the interface. (C) Residues involved in the hydrophobic interaction are shown as sticks.

Fig. 2.

Induced-fit binding between Brd4 ET domain and the MLV IN EBM. (A) Isothermal titration calorimetry of the ET–EBM interaction reveals high affinity, with K_d 159 ± 12 nM. (B) ¹H-¹⁵N–correlation spectra of [U-¹⁵N]-Brd4 ET domain in the absence (black) and presence of unlabeled MLV IN EBM (red). Resonances with large chemical shift perturbations (CSPs) are labeled with their amino acid assignment. (All backbone amide assignments and plot of CSPs are shown in Fig. S2.) (C) One-dimensional ¹H spectrum of the MLV EBM free (black) and bound to the ET domain, with protein signals suppressed by a [¹³C, ¹⁵N] filter (blue). Peaks labels indicate well-dispersed signals in the bound peptide, blue labels for backbone signals and green for side-chain signals.

Fig. 3.

Solution structure of the complex between Brd4 ET and MLV IN EBM. (A) Ensemble of 20 lowest-energy structures of the complex between the Brd4 ET domain (gray) and EBM (yellow). (B) Ribbon diagram of the lowest-energy conformer; the new β sheet comprises antiparallel strands β1 from the ET domain (residues 650–654) and β6′ (391′–395′) and β7′ (400′–404′) from the EBM (Fig. S3). The protein ribbon is colored according to backbone amide shift perturbations (as quantified in Fig. S2). (C and D) Electrostatic potential map of the binding interface between Brd4 ET and the EBM, illustrating charged and hydrophobic residues in the intermolecular interface (Fig. 4).

Fig. S2.

Chemical shift perturbations on Brd4 from MLV IN EBM binding. (A) ¹H-¹⁵N–correlation spectra of [U-¹³C, ¹⁵N] - Brd4 ET in the absence (black) and presence (red) of MLV IN EBM. All peaks are labeled with blue lines indicating perturbations. (B) Bar graph of EBM-induced chemical shift perturbation (CSP) values calculated according to ${\mathrm{\Delta }\delta }_{ppm}=\sqrt{0.5\left(\mathrm{\Delta }\delta H^2+\left(\mathrm{\Delta }\delta N^2/25\right)\right)}$. Bar on the right indicated the color mapped to the structure. (C) The CSPs mapped to the structure. The MLV IN EBM is colored yellow.

Fig. S3.

NMR-based evidence for β sheet formation. (A) Zoomed region of the 2D ¹³C/¹⁵N-filtered NOESY spectrum showing HN and HA. Labels show peaks for intraresidue HN-to-HA peaks. Red lines indicate NOESY walk for peptide assignments. Blue letters are characteristic NOESY peaks for interstrand β sheet. (B) Schematic diagram of backbone residues involved in the three-stranded β sheet. Red dashed lines are H-bonds, blue lines are observed interstrand NOEs, and blue dashed lines are expected NOEs that are not positively identified due to spectral overlap. Letters correspond to peaks labeled in A and C. (C) Alternating strips of the 3D ¹⁵N-edited NOESY and 3D ¹³C/¹⁵N-filtered (f1) ¹⁵N-edited NOESY (f3) for residues I652 and I654 within the β sheet. NOEs are assigned; blue letters correspond to interstrand NOEs.

Fig. 4.

Specific side-chain interactions between Brd4 ET and MLV IN EBM. (A) Complementary arrangement of positively charged side chains on β7′ of IN (blue) and negatively charged side chains of the ET domain (black); ET domain residues mutated for binding studies are labeled in red. (B) Hydrophobic interface defined by conserved nonpolar side chains on Brd4 and MLV IN. Label colors as in A. (C) Affinity capture assay shows that MLV IN binds to WT FLAG-tagged Brd4 (1–720) (lane 4), but not mutants designed to disrupt the ET–EBM interface, V634S/I652S and E653R/D655R (lanes 5 and 6). No Brd4 is detected bound to the resin in the absence of GST-tagged MLV-IN (lanes 1–3); lanes 7–9 are immunoblots of 1/10th of the protein loaded in lanes 1–6. (D) The His-tagged Brd4 ET domain can bind NSD3 in the absence (lane 3), but not in the presence, of the EBM peptide (lane 2), demonstrating competition between the viral and host factors for ET binding. Lane 1 shows the presence of NSD3 in the extract, lane 4 shows no cross-reactivity, and lane 5 shows that NSD3 is not retained on the Ni resin in the absence of Brd4. (E) The FLAG-tagged Brd4 can bind NSD3 in the absence (lane 1), but not in the presence, of the EBM peptide (lane 2), demonstrating competition between the viral and host factors for ET binding. Lane 3 shows that NSD3 is not retained on the Ni resin in the absence of Brd4.

Fig. S4.

Ectopic expression profiles of Brd4 mutants in HEK293T cells. Affinity capture assays show that WT FLAG-Brd4 (1–720) (lanes 2 and 5) had expression levels similar to those of FLAG-Brd4 V634S/I652S (lanes 3 and 6) and FLAG-Brd4 E653R/D655R (lanes 4 and 7). (A) A Coomassie gel of the resulting proteins from an affinity capture assay using 1 mg total cellular protein. Molecular weight standards are indicated. (B) Anti-FLAG Western blot with 80 µg total cellular protein used per each pull-down assay. Also indicated to the right are the heavy (HC) and light (LC) chains of the anti-FLAG beads.