Structure of a BCOR corepressor peptide in complex with the BCL6 BTB domain dimer

Abstract

The transcriptional corepressors BCOR, SMRT, and NCoR are known to bind competitively to the BCL6 BTB domain despite the fact that BCOR has no detectable sequence similarity to the other two corepressors. We have identified a 17 residue motif from BCOR that binds directly to the BCL6 BTB domain and determined the crystal structure of the complex to a resolution of 2.6 A. Remarkably, the BCOR BCL6 binding domain (BCOR(BBD)) peptide binds in the same BCL6 binding site as the SMRT(BBD) peptide despite the lack of any significant sequence similarity between the two peptides. Mutations of critical BCOR(BBD) residues cause the disruption of the BCL6 corepression activities of BCOR, and a BCOR(BBD) peptide blocks BCL6-mediated transcriptional repression and kills lymphoma cells.

Figure 1. Identification of the BCOR BCL6 Binding Domain (BCOR^BBD)

(A) BCOR fragments were purified as thioredoxin (Trx) fusion proteins and assayed for binding to the BCL6 BTB domain by native gel electrophoresis. The horizontal box indicates the position of unbound BCL6 BTB. BCL6^BTB was loaded alone (lane “-“) or as a mixture with the Trx-BCOR fragments described in panel B. Equal amounts of BCL6^BTB were loaded in all lanes. (B) Schematic of the long-isoform BCOR protein, indicating the fragments used in the binding assay in panel (A). (C) Crystal structure of the BCL6^BTB/BCOR^BBD complex. The BCL6^BTB dimer is shown in cartoon representation with blue and pink subunits. The two bound BCOR^BBD peptides are shown in bond representation with green and yellow carbons, respectively.

Figure 2. Comparison of the BCOR and SMRT BBD complex structures

(A) View of the lateral binding groove in the BCL6^BTB/BCOR^BBD complex. The BTB dimers are shown as solvent accessible surfaces with subunit coloring as in Figure 1C. The asterisk indicates the position of residue H116 from BCL6, which is buried beneath the BBD. (B) Superposition of BCOR^BBD (green) and SMRT^BBD (red) peptides, represented as Cα traces with added Cβ positions. The boxed region indicates BCOR residues A505-S508 and SMRT residues G1422-I1425, in which the BBDs adopt significantly different conformations. (C) The BCL6^BTB/SMRT^BBD complex (Ahmad et al., 2003). In this case, BCL6 residue H116 (asterix) covers SMRT^BBD residues I1425 and S1424. (D) Expanded view of the central region of the superposed BCOR and SMRT BBDs. (E) Structure-based sequence alignment of the BCOR and SMRT BBDs. Grey shading indicates the four residues that form similar side chain contacts with the BCL6 BTB domain. These are the only positions with sequence similarity (I/V; W/H) or identity (Pro) between the BCOR and SMRT BBDs. The graph shows the variation in the average Cα position between the BCOR and SMRT peptides. The error bars are the standard deviation of the 16 independent measured distances, based on two independent SMRT and eight independent BCOR crystallographic observations of the peptide structures.

Figure 3. Contacts between BCL6 and the SMRT and BCOR BBDs

(A) Schematic representation of conserved BCL6 contacts in the BCOR and SMRT BBDs. The complete BBD sequences are shown, while only the BCL6 residues involved in BBD contacts are shown. Green and red ovals highlight the residues participating in main chain and side chain contacts, respectively. Black ovals enclose the residues not forming conserved contacts. A conserved water at the peptide-protein interface near BCL6 Arg13 is indicated as an encircled “W”. Most of the contacts are polar and involve main chain atoms from the BBD peptides. (B) Similar to panel (A), but showing non-conserved interactions. In this case, most of the contacts are non-polar van der Waal packing interactions involving BBD side chains.

Figure 4. Alanine scan of the BCOR and SMRT corepressor peptides

(A) Fluorescence Polarization (FP) binding curve for A488-SMRT as a function of the BCL6^BTB concentration. (B) Representative competition binding curves in which unlabelled peptides are added to disrupt the BCL6^BTB/A488-SMRT complex. Each residue in BCOR^BBD or SMRT^BBD was replaced by an alanine (or glycine where the native residue is an alanine) in a series of non-fluorescent synthetic peptides. Triangles: SMRT^BBD, squares BCOR^BBD, circles: BCOR^BBD W509A. The complete set of titrations are included in Figures S7 and S8. (C) The Ki values (μM) derived from the FP competition curves for the single residue Ala or Gly substituted BCOR^BBD and SMRT^BBD peptides are indicated below a plot of the average buried surface area of the residues of the BCOR^BBD (green) and SMRT^BBD (red) peptides in the crystal structure.

Figure 5. Mutation of the BCOR BBD specifically affects its ability to function with BCL6

(A) BCOR BBD mutations abrogate the interaction of BCOR with BCL6 in vivo. HEK293 cells were transfected with 0.1 μg each of BCL6 expression plasmid alone or together with 0.5 μg myc-BCOR or myc-BCOR-mt (BCOR S507A/W509A/V511A mutant). Cell lysates were immunoprecipitated with α-myc antibody and recovered proteins were detected by western blotting using N-3 α-BCL6 and 9E10 α-myc antibodies. (B) Mutation of the BCOR BBD abrogates the ability of BCOR to potentiate BCL6 repression. (C) Mutation of the BCOR BBD does not affect the ability of BCOR to repress AF9 activation. (D) BCL6 positive (Ly1, 7 and 10) and BCL6 negative (Ly4) diffuse large B-cell lymphoma cell lines where exposed to 5 μM TAT-BCOR^BBD or a control peptide. Levels of BCL6 target genes CD69, p21 and CD80 as well as the negative control (non-BCL6 target gene) CD20 were measured by QPCR. (E) Ly1, 4, 7 and 10 cells were allowed to grow for 48 hours in the presence of no added BCOR inhibitor peptide (black bars); 2 μM peptide (grey bars); or 5 μM peptide (open bars). Error bars in panels B-E indicate the standard error.