A common binding motif in the ET domain of BRD3 forms polymorphic structural interfaces with host and viral proteins

Abstract

The extraterminal (ET) domain of BRD3 is conserved among BET proteins (BRD2, BRD3, BRD4), interacting with multiple host and viral protein-protein networks. Solution NMR structures of complexes formed between the BRD3 ET domain and either the 79-residue murine leukemia virus integrase (IN) C-terminal domain (IN_329-408) or its 22-residue IN tail peptide (IN_386-407) alone reveal similar intermolecular three-stranded β-sheet formations. ¹⁵N relaxation studies reveal a 10-residue linker region (IN_379-388) tethering the SH3 domain (IN_329-378) to the ET-binding motif (IN_389-405):ET complex. This linker has restricted flexibility, affecting its potential range of orientations in the IN:nucleosome complex. The complex of the ET-binding peptide of the host NSD3 protein (NSD3_148-184) and the BRD3 ET domain includes a similar three-stranded β-sheet interaction, but the orientation of the β hairpin is flipped compared with the two IN:ET complexes. These studies expand our understanding of molecular recognition polymorphism in complexes of ET-binding motifs with viral and host proteins.

Keywords: BET proteins; BRD3; Moloney murine leukemia virus; NSD3; extraterminal domain; integrase; interdomain dynamics; isotope peptide labeling; solution NMR.

Fig. 1.. Domain and motif constructs used in this study.

The full-length proteins are indicated, with the domains utilized in this study highlighted in red and well-annotated domains indicated in blue. BRD3_1–726 consists of two bromodomains (BD1 and BD2) and the extra terminal (ET) domain. MLV IN_1–408 consists of the N-terminal region (NTR), catalytic core domain (CCD) and the C-terminal domain (CTD). The sequence of the 22-residue tail peptide (TP) is shown. NSD3 consists of the ET binding region (EBR), two PWWP domains (P), an AWS domain (A), a SET domain, and a post-SET domain.

Fig. 2.. Solution NMR structure of BRD3 ET.

(A) Primary sequence of the BRD3 ET construct, including the His₆ tag. Numbering starts with the BRD3 G554. (B) Representation of the backbone structure of the entire BRD3 ET domain (residues 554–640), including the N-terminal His₆ tag. Disordered region including the N-terminal His₆ tag is represented in magenta. Further analysis was performed with respect to only the ordered region (blue). (C) Ribbon representation of the ordered region of BRD3 ET domain (569–640). The 3 α-helices are α1: 574–586, α2: 590–602, and α3: 624–637. The ordered region of the protein includes residues 569–640. (D) Surface electrostatic representation of the ordered region of BRD3 ET. Fully saturated red and blue colors represent, respectively, negative and positive potentials of ± 5 kT at an ionic strength of 0.15 M at 298 K. Key negatively charged residues and residues forming the acidic cluster (D612-E619) are marked. (E) Surface representation of hydrophobic residues of BRD3 ET reveals the presence of a hydrophobic pocket. Hydrophobicity ranges from brown to white as least hydrophobic residues based on hydrophobicity scale (Eisenberg et al., 1984).

Fig. 3.. Interaction of MLV IN TP with BRD3 ET.

(A) Schematic of the primary sequence of the MLV IN TP and BRD3 ET regions utilized in the studies, together with the NMR defined secondary structural elements. The N-terminal BRD3 ET His₆ tag is not shown. (B-D) Surface hydrophobic representations of the complex with (B) and (C) oriented in the same plane and (D) rotated along the y axis by 180°. (B) is set at 50% transparency and also shows a ribbon representation of the complex with blue/magenta showing BRD3 ET and cyan showing the MLV IN TP. The surface hydrophobicity coloring scheme is the same as in Figure 2E. (C) is the buried face of the BRD3 ET and (D) the buried face of the MLV IN TP. Key residues forming the hydrophobic pocket are indicated. (E-G) Surface electrostatic representation (coloring scheme same as Figure 2D) with (E) BRD3 ET and IN TP complex, (F) BRD3 ET and (G) MLV IN TP having identical viewing planes as in (B-D). Key residues forming intermolecular electrostatic interactions are indicated.

Fig. 4.. Linker flexibility between SH3 fold and TP of IN CTD in complex with ET.

(A) Schematic of the primary sequence and NMR defined secondary structure of the MLV IN CTD and BRD3 ET regions utilized in these studies. The SH3 fold is shown in orange, the linker between SH3 fold and TP in yellow, the structured region of the ETBM in red, and the ET domain in green. Transition residues between the SH3 / linker and linker / ETBM are boxed (see Fig. 5D). (B) Ribbon representation of the full CTD-ET complex with color scheme described in A. The ET-peptide structure (blue / magenta / cyan) is overlaid onto the full complex. (C) Backbone representation of the NMR ensemble for the complex, superimposed on the CTD SH3 fold region. (D) Backbone representation of the NMR ensemble for the complex, superimposed on the ETBM : ET domain. Coloring scheme in panels C and D is the same as in panel B. Non-native tags and the disordered ET region (residues 554–568) are not shown for clarity.

Fig. 5.. Analysis of chemical shift perturbations and ¹⁵N-¹H heteronuclear NOE data for the MLV IN CTD : BRD3 ET complex.

(A) Secondary structure features correlating with the amino-acid residue positions in the X-axis in panel B and C. (//) indicates the junction between the C-terminus of the IN CTD and the N-terminus of the BRD3 ET domain. Numbering corresponds to the individual domains in the corresponding full-length protein sequences. Domain/motif coloring scheme: Orange, MLV IN CTD SH3 fold; Yellow, MLV IN CTD linker region; Red, MLV IN CTD ETBM; Green, BRD3 ET domain. (B) N-H bond rotational correlation time measurements (τ_c in ns) for each residue across the MLV IN CTD and BRD3 ET complex. Purple horizontal line, overall τ_c (4.1 ns) expected for freely-mobile SH3 domain (10.1 kDa) at 25 °C. Blue horizontal line, overall τ_c (8.4 ns) expected for freely-mobile ETBM : BRD3 ET complex (14.2 kDa) at 25 °C; Black horizontal line, overall τ_c (13.3 ns) expected for rigid MLV IN CTD : ET complex (21 kDa), at 25 °C. Uncertainties of these estimates, determined as described in the STAR methods section, are indicated with error bars. (C) ¹H-¹⁵N HetNOE signal (I_saturated/I_equilibrium) calculated for each residue across the complex. Uncertainties of these estimates, determined as described in the STAR methods section, are indicated with error bars. (D) Summary of motif boundaries. The sequence of the MLV IN TP used in the NMR structural studies is indicated (top). The SH3 fold (orange) transitions into the linker region at Ala377 and Ala378, which maintain higher HetNOEs compared to their τ_c values. The ten amino-acid residues within the Linker region are indicated in yellow. Thr389 transitions the Linker into the ETBM (red). Residues labeled with * correspond to Prolines; no values are plotted for residues that were not assigned, with poor signal-to-noise ratios, and/or with poor relaxation curve fits.

Fig. 6.. Interaction of BRD3 ET with NSD3_148–184 peptide.

(A) Schematic of the primary sequence with NMR defined secondary structure of the NSD3_148–184 and BRD3 ET (N-terminal BRD3 ET His₆ tag is not shown) utilized in the studies. (B-D) Surface hydrophobic representations of the complex with (B) and (C) oriented in the same plane and (D) rotated along the y axis by 180°. (B) is set at 50% transparency and also shows a ribbon representation of the complex with blue/magenta showing BRD3 ET and green showing the NSD3_148–184. The surface hydrophobicity coloring scheme is the same as in Figure 2E. (C) is the buried surface of the BRD3 ET and (D) is that of the NSD3_148–184. Key residues forming the hydrophobic pocket are indicated. (E-G) Surface electrostatic representations (coloring scheme same as Figure 2D) of (E) the BRD3 ET-NSD3_148–184 complex, (F) BRD3 ET and (G) NSD3_148–184 having identical viewing planes as in (B-D). Key residues forming the electrostatic interactions are indicated.

Fig. 7.. Comparison of peptide : ET complex structures.

Coloring of ET domain in complex with the MLV IN TP and NSD3 peptide is as described in Figures 3 and 6. (A and B) (A) BRD3 ET (blue)-MLV IN TP (cyan) and (B) BRD3 ET (blue)-NSD3_148–184 (green). (C) Space filling model of BRD3 ET (beige) overlayed with TP (cyan) and NSD3_148–184 (green). (D) Side-chain orientation of key interacting residues of the TP (cyan) with NSD3_148–184 (green). (E) Comparison of the single β strand formed using the short NSD3 peptide 152–163 (PDB: 2NCZ) in black and the alignment with the β strand formed in the ET domain (black), with the two β strands formed in the BRD3 ET NSD3_148–184 complex in green and the alignment of the β strand of ET in magenta. (F) Side-chain orientations of key interacting residues of the NSD3_152–163 (black) with NSD3_148–184 (green). (G) Alignment of key interacting residues of BRD3 ET domain EIEID (black) forming antiparallel β strand with TP (blue) and NSD3_148–184 peptide (green).

Fig. 8.. Intasome overlay.

Left panel represents the overlay of PFV intasome with the MLV IN CTD and BRD3 ET complex (this study). The alignment of PFV intasome-IN CTD:ET complex onto nucleosome was guided using the PFV intasome-nucleosome cryo-EM structure (PDB ID 6RNY; EMDB ID 4960) (Wilson et al., 2019). The PFV intasome dimer of dimers is represented in yellow and blue color, the IN CTD is in orange, BRD3 ET complex is in green, the viral and target DNA is in magenta and core histone proteins are in grey. Right panel is a 90° rotation along the y-axis.