Reactivation of mutant p53 through interaction of a C-terminal peptide with the core domain

Abstract

A synthetic 22-mer peptide (peptide 46) derived from the p53 C-terminal domain can restore the growth suppressor function of mutant p53 proteins in human tumor cells (G. Selivanova et al., Nat. Med. 3:632-638, 1997). Here we demonstrate that peptide 46 binds mutant p53. Peptide 46 binding sites were found within both the core and C-terminal domains of p53. Lys residues within the peptide were critical for both p53 activation and core domain binding. The sequence-specific DNA binding of isolated tumor-derived mutant p53 core domains was restored by a C-terminal polypeptide. Our results indicate that C-terminal peptide binding to the core domain activates p53 through displacement of the negative regulatory C-terminal domain. Furthermore, stabilization of the core domain structure and/or establishment of novel DNA contacts may contribute to the reactivation of mutant p53. These findings should facilitate the design of p53-reactivating drugs for cancer therapy.

FIG. 1

Peptide 46 binds to the His-273 mutant p53 protein in a cellular extract. (A) Biotinylated peptide 46 and its mutant derivative peptide A3, carrying Ile substitutions for Lys residues 370, 372, and 373, were immobilized on streptavidin-coated Dynabeads and used for precipitation of p53 from an SW480 cell extract. Bound proteins were analyzed by Western blotting using the p53-specific monoclonal antibody DO-1. Peptide 46 precipitated mutant p53 protein from the SW480 cell extract (lane 2), whereas no p53 was precipitated by control peptide A3 (lane 3) or Dynabeads without peptide (lane 4). Lane 1, 5% of input of cell lysate. (B) Western blot analysis of p53 precipitated from Saos-2-His-273 cells grown in the absence (p53 on; lanes 1 to 4) or presence (p53 off; lanes 5 to 8) of doxycycline. Peptide 46 (lane 4), but neither the mutant control peptide A3 (lane 2), peptide A4 that carries Ile substitutions for Lys residues 381 and 382 (lane 3), nor unloaded Dynabeads (not shown), precipitated p53. Lanes 1 and 5, 5% of input of lysate from cells grown in the absence and presence of doxycycline, respectively. Experimental conditions were as for panel A.

FIG. 2

Peptide 46 interacts with the core and C-terminal domains of p53. (A) Biotinylated peptide 46 or its mutant derivative peptides A3 and A4, carrying Ile substitutions for Lys 370, 372, and 373 and of Lys 381 and 382, respectively, were immobilized on streptavidin-coated Dynabeads and incubated with p53 deletion mutant proteins representing the N-terminal [GST-p53(1-100)], core [GST-p53(99-307)], and C-terminal [GST-p53(320-393)] domains. Bound proteins were eluted and analyzed by Western blotting using the p53-specific monoclonal antibodies PAb421 (top), PAb240 (middle), and DO-1 (bottom) to detect the C-terminal, core, and N-terminal domain polypeptides, respectively. (B) The ³²P-labeled GST-p53(99-307) core domain protein was incubated with increasing amounts (100 and 200 ng) of the GST-p53(320-393) C-terminal domain protein (lanes 2 to 5, upper panel), 200 ng of the GST-p53(1-100) N-terminal domain protein (lane 6, upper panel), or a series of overlapping synthetic peptides spanning the p53 C terminus (lower panel). The protein-protein and peptide-protein complexes were separated by native polyacrylamide gel electrophoresis and visualized by autoradiography.

FIG. 3

Lysine residues at positions 370, 372, 373, 381, and 382 are critical for both p53 activation and core domain binding by peptide 46. (A) Plasmid PG-CAT was cotransfected with a wild-type p53 expression plasmid and a series of mutant derivatives of peptide 46. Ala substitution for Lys residues at positions 370, 372, 381, and 382 in peptide S33 or for residues at positions 363, 365, 368, 370, 372, and 373 in peptide S35 abolished the ability of peptide 46 to stimulate p53-mediated CAT reporter gene activation (columns 6 and 7), whereas the single Ala substitutions in peptides S21 (Lys 370), S22 (Ser 371), and S30 (His 380) had little or no effect (columns 3 to 5). (B) Mutant derivatives of peptide 46 were tested for the ability to activate the specific DNA binding of baculovirus- produced p53 in a band shift assay as described in Materials and Methods. Peptides S21, S22, and S30, carrying single Ala substitutions, were able to stimulate specific DNA binding of p53, although with variable efficiency, whereas peptides S33 and S35, carrying Ala substitutions for several Lys residues, were inactive. The sequence specificity of p53 DNA binding induced by the peptides was demonstrated by competition of p53-DNA complexing with specific (S) oligonucleotide BC (lanes 3, 6, 9, and 12) but not with nonspecific (N) control oligonucleotide MN (lanes 4, 7, 10, and 13). (C) The ³²P-labeled GST-p53(99-307) core domain protein was incubated with synthetic peptides and analyzed in a native gel mobility shift assay as described for Fig. 2B. Peptides 46, S21, S22, and S30, but not peptides S33, S35, A3, and A4, caused a shift in the migration of the core domain protein on the native gel. (D) GST–full-length p53 proteins carrying Ile substitutions for Lys residues at positions 370, 372, and 373 (I370/372/373; lanes 2 to 5) and 381, 382, and 386 (I381/382/386; lanes 6 to 9) were tested for specific DNA binding in a band shift assay as described for panel B. Lane 1, GST–wild-type (WT) p53 protein. The specific oligonucleotide BC (S) competed out p53-DNA complexes (lanes 3 and 7), whereas the nonspecific (N) control oligonucleotide MN with either blunt (lanes 4 and 8) or protruding (lanes 5 and 9) ends did not compete.

FIG. 4

The isolated wild-type core domain binds DNA more efficiently upon complexing with the C-terminal polypeptide. (A) Interaction of the p53 C-terminal domain protein (residues 320 to 393) with the core domain protein (residues 99 to 307) stimulated the sequence-specific DNA binding of the core domain in a band shift assay (lanes 1 and 2). Addition of the GST-p53(320-393) protein induced the formation of a new protein-DNA complex, complex II (lane 2), in a dose-dependent manner (lanes 8 to 10). Complex II was efficiently competed by a two- or fivefold excess of the specific (S) oligonucleotide BC (lanes 3 and 4) but not by the nonspecific control (contr) oligonucleotide MN (lanes 5 and 6). The GST-p53(320-393) protein alone did not bind DNA under the conditions used (lane 7). (B) The C-terminal domain protein is present in a core domain-DNA complex, as determined by Western blot analysis using the monoclonal antibodies PAb240 (upper panel) and PAb421 (lower panel) to detect proteins eluted from the region of a preparative gel corresponding to complex II (lanes 1, 3, and 5) and complex I (lanes 2, 4, and 6) after incubation of DNA with the C-terminal domain protein only (lanes 1 and 2), both the C-terminal and core domain proteins (lanes 3 and 4), and the core domain protein only (lanes 5 and 6). Lane 7, control GST-p53(320-393) protein alone; lane 8, control GST-p53(99-307) protein alone. (C) The GST-p53Δ30 protein, which lacks the 30 C-terminal amino acid residues and is constitutively activated for specific DNA binding, was further activated by peptide 46 in a band shift assay (lanes 1 and 3). Lane 1, GST-p53Δ30 in the absence of peptide 46; lane 2, GST-p53Δ30 in the presence of the activating antibody PAb421; lane 3, GST-p53Δ30 in the presence of peptide 46. The GST-p53Δ30-DNA complex induced by peptide 46 was competed by the specific (S) oligonucleotide BC (lane 4) but not by the nonspecific (N) control oligonucleotide MN (lane 5).

FIG. 5

The sequence-specific DNA binding of isolated mutant core domains is restored by the C-terminal polypeptide. (A) The wild-type (wt) GST-p53(99-307) protein bound to the labeled specific BC oligonucleotide in a band shift assay (lane 1). The core domain-DNA complex was competed by a 10-fold excess of the specific (S) oligonucleotide BC (lane 2) but not by a 10-fold excess of the nonspecific (N) MN oligonucleotide (lane 3). GST-p53(99-307) proteins carrying tumor-derived mutations His-273 (B), Trp-248 (D), His-175 (D), Ala-143 (E), and Ser-249 (F) did not bind the labeled BC oligonucleotide in the absence of the C-terminal domain polypeptide (lanes 1) but formed a complex with BC in the presence of the C-terminal polypeptide (lanes 2). The DNA-mutant core domain complexes were competed out by a 10-fold excess of the unlabeled specific (S) oligonucleotide BC (lane 3) but not by the nonspecific (N) oligonucleotide MN (lane 4).