Design of potent inhibitors of human RAD51 recombinase based on BRC motifs of BRCA2 protein: modeling and experimental validation of a chimera peptide

Abstract

We have previously shown that a 28-amino acid peptide derived from the BRC4 motif of BRCA2 tumor suppressor inhibits selectively human RAD51 recombinase (HsRad51). With the aim of designing better inhibitors for cancer treatment, we combined an in silico docking approach with in vitro biochemical testing to construct a highly efficient chimera peptide from eight existing human BRC motifs. We built a molecular model of all BRC motifs complexed with HsRad51 based on the crystal structure of the BRC4 motif-HsRad51 complex, computed the interaction energy of each residue in each BRC motif, and selected the best amino acid residue at each binding position. This analysis enabled us to propose four amino acid substitutions in the BRC4 motif. Three of these increased the inhibitory effect in vitro, and this effect was found to be additive. We thus obtained a peptide that is about 10 times more efficient in inhibiting HsRad51-ssDNA complex formation than the original peptide.

Figure 1

Interaction interface between the BRC4 peptide and HsRad51 protein. The BRC4 peptide (ribbon)/HsRad51 (mesh) interface is visualized from their complex structure.(21) The strong contact zones are noted in the 3-D structure and on the 28-amino acid sequence. The secondary structure of the peptide is indicated beside the sequence.

Figure 2

Effect of deletions in the N- and C-terminal parts of the BRC4 peptide on its capacity to dissociate the HsRad51−DNA complex. The dissociation of the HsRad51−ssDNA complex by N-terminal (A) and C-terminal deleted (B) BRC4-motif peptides was monitored by the decrease in fluorescence intensity of poly(dεA) upon stepwise addition of the peptides: BRC4-28 (closed diamonds), BRC4-26 (closed circles), BRC4-24c (closed squares), BRC4-24 (stars), and BRC4-24n (open squares). The amino acid sequence of peptides is noted under the figure to facilitate the visualization of the deleted parts. The experiments were performed using 1.5 μM HsRad51 and 3 μM (in bases) poly(dεA) in the presence of 1 mM ATP.

Figure 3

α-Helical folding potential of BRC peptides: CD analysis. CD spectra of 40 μM BRC peptides were measured in the presence (lower parts) and absence (upper parts) of 1 mM SDS. (A) CD of deleted peptides (BRC4-24, black; BRC4-24n, orange; BRC4-24c, cyan). (B) Substituted peptides (H/Y, magenta; A/S₁, olive; A/S₂, purple; L/F, navy).

Figure 4

Proposition of amino acid substitutions of the BRC4 peptide to gain in interaction energy by comparison with the interaction of other BRC motifs with HsRad51. In (A), the amino acid sequences of the eight human BRC motifs are aligned and the best residues in interaction energy to HsRad51 at the given position are colored. In (B), the interaction energy of residues of different BRC motifs at given positions is shown. In (C), relative local energetic gain (ΔEi) is calculated for each substitution. The secondary structure of the BRC4 motif is presented at the left side to facilitate the identification of substitution positions.

Figure 5

Effect of amino acid substitutions on the contact with HsRad51. (A) Substitution of Ala1535 by serine creates a new hydrogen bonding with Glu213 of HsRad51. (B) Substitution of L1545 by phenylalanine strengthens the interaction with a hydrophobic pocket of HsRad51 (gray surface).

Figure 6

Effect of amino acid substitutions of the peptide on its capacity to dissociate the HsRad51−DNA complex. The dissociation of the HsRad51−ssDNA complex by modified BRC4-motif peptides was monitored by the decrease in fluorescence intensity of poly(dεA) upon stepwise addition of the peptides as in Figure 2. Open diamonds, A/S₁ (Ala1535Ser); open circles, H/Y (His1525Tyr); closed squares, L/F (Leu1545Phe); crosses, A/S₂ (Ala1535Ser); open triangles, Y-S₂-F peptide with three amino acid substitutions.

Figure 7

Electron microscopy observation of the effect of Y-S₂-F BRC peptide on the HsRad51−ssDNA complexes. HsRad51−ssDNA filaments formed between HsRad51 (5 μM) and ΦX174 ssDNA (10 μM in bases) were observed by electron microscopy after addition of 10 μM (B) or 50 μM (C) Y-S₂-F BRC peptide or without peptide (A).

Figure 8

Inhibition of the DNA strand exchange reaction by the modified BRC4 peptides. The HsRad51-promoted (0.5 μM) strand exchange reaction between single-stranded and double-stranded oligonucleotides in the presence of indicated concentrations of BRC4 peptides was analyzed by quantifying the reaction product (B) after its separation by polyacrylamide gel electrophoresis (A) as described in the Experimental Section. Stars, BRC4-24; circles, H/Y; squares, L/F; crosses, A/S2; triangles, Y-S2-F peptides.