A Stable Human p53 Heterotetramer Based on Constructive Charge Interactions within the Tetramerization Domain
2003; Elsevier BV; Volume: 278; Issue: 4 Linguagem: Inglês
10.1074/jbc.m208528200
ISSN1083-351X
AutoresRichard D. Brokx, Eleonora Bolewska‐Pedyczak, Jean Gariépy,
Tópico(s)Cancer Research and Treatments
ResumoThe human p53 tetramerization domain (called p53tet; residues 325–355) spontaneously forms a dimer of dimers in solution. Hydrophobic interactions play a major role in stabilizing the p53 tetramer. However, the distinctive arrangement of charged residues at the dimer-dimer interface suggests that they also contribute to tetramer stability. Charge-reversal mutations at positions 343, 346, and 351 within the dimer-dimer interface were thus introduced into p53tet constructs and shown to result in the selective formation of a stable heterotetramer composed of homodimers. More precisely, mutants p53tet-E343K/E346K and p53tet-K351E preferentially associated with each other, but not with wild-type p53tet, to form a heterodimeric tetramer with enhanced thermal stability relative to either of the two components in isolation. The p53tet-E343K/E346K mutant alone assembled into a weakly stable tetramer in solution, whereas p53tet-K351E existed only as a dimer. Moreover, these mutants did not form heterocomplexes with wild-type p53tet, illustrating the specificity of the ionic interactions that form the novel heterotetramer. This study demonstrates the dramatic importance of ionic interactions in altering the stability of the p53 tetramer and in selectively creating heterotetramers of this protein scaffold. The human p53 tetramerization domain (called p53tet; residues 325–355) spontaneously forms a dimer of dimers in solution. Hydrophobic interactions play a major role in stabilizing the p53 tetramer. However, the distinctive arrangement of charged residues at the dimer-dimer interface suggests that they also contribute to tetramer stability. Charge-reversal mutations at positions 343, 346, and 351 within the dimer-dimer interface were thus introduced into p53tet constructs and shown to result in the selective formation of a stable heterotetramer composed of homodimers. More precisely, mutants p53tet-E343K/E346K and p53tet-K351E preferentially associated with each other, but not with wild-type p53tet, to form a heterodimeric tetramer with enhanced thermal stability relative to either of the two components in isolation. The p53tet-E343K/E346K mutant alone assembled into a weakly stable tetramer in solution, whereas p53tet-K351E existed only as a dimer. Moreover, these mutants did not form heterocomplexes with wild-type p53tet, illustrating the specificity of the ionic interactions that form the novel heterotetramer. This study demonstrates the dramatic importance of ionic interactions in altering the stability of the p53 tetramer and in selectively creating heterotetramers of this protein scaffold. matrix-assisted laser desorption ionization time-of-flight size-exclusion chromatography wild-type Human p53 plays an important role in tumor suppression (1Lane D.P. Nature. 1992; 358: 15-16Crossref PubMed Scopus (4437) Google Scholar, 2Arrowsmith C.H. Morin P. Oncogene. 1996; 12: 1379-1385PubMed Google Scholar, 3Bargonetti J. Manfredi J.J. Curr. Opin. Oncol. 2002; 14: 86-91Crossref PubMed Scopus (311) Google Scholar, 4Somasundaram K. Front. Biosci. 2000; 5: D424-D437Crossref PubMed Google Scholar). It is a modular protein consisting of discrete functional domains, which can be expressed and studied in isolation. In particular, residues 325–355 of human p53 (p53tet) spontaneously form a tetramer in solution (see Fig. 1 a) (5Chene P. Oncogene. 2001; 20: 2611-2617Crossref PubMed Scopus (202) Google Scholar, 6Jeffrey P.D. Gorina S. Pavletich N.P. Science. 1995; 267: 1498-1502Crossref PubMed Scopus (435) Google Scholar, 7Lee W. Harvey T.S. Yin Y. Yau P. Litchfield D. Arrowsmith C.H. Nat. Struct. Biol. 1994; 1: 877-890Crossref PubMed Scopus (234) Google Scholar). Each monomer within the context of the p53tet domain adopts an identical structure,viz. a short N-terminal β-strand (residues 326–333) followed by a turn and a C-terminal α-helical domain (residues 335–354). Two monomers associate in an antiparallel fashion through contacts between β-sheet strands as well as hydrophobic interactions involving α-helical residues to form a "primary dimer" (6Jeffrey P.D. Gorina S. Pavletich N.P. Science. 1995; 267: 1498-1502Crossref PubMed Scopus (435) Google Scholar, 7Lee W. Harvey T.S. Yin Y. Yau P. Litchfield D. Arrowsmith C.H. Nat. Struct. Biol. 1994; 1: 877-890Crossref PubMed Scopus (234) Google Scholar). One significant salt bridge in the p53tet region occurs between Arg337 of one subunit and Asp352 of its adjacent subunit (side chain oxygen–nitrogen distance of 2.72 Å) (6Jeffrey P.D. Gorina S. Pavletich N.P. Science. 1995; 267: 1498-1502Crossref PubMed Scopus (435) Google Scholar), stabilizing the structure of the primary dimer (8Davison T.S. Yin P. Nie E. Kay C. Arrowsmith C.H. Oncogene. 1998; 17: 651-656Crossref PubMed Scopus (94) Google Scholar, 9DiGiammarino E.L. Lee A.S. Cadwell C. Zhang W. Bothner B. Ribeiro R.C. Zambetti G. Kriwacki R.W. Nat. Struct. Biol. 2002; 9: 12-16Crossref PubMed Scopus (213) Google Scholar). Two primary dimers then self-associate through an interface derived from residues located in their α-helical domains to form a "dimer of dimers," referred to as a p53 tetramer. Mutations of amino acids at this interface have highlighted the importance of hydrophobic residues leading to the formation of the tetramer as well as stable p53 dimers (10Mateu M.G. Fersht A.R. EMBO J. 1998; 17: 2748-2758Crossref PubMed Scopus (126) Google Scholar, 11Mateu M.G. Fersht A.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3595-3599Crossref PubMed Scopus (59) Google Scholar, 12McCoy M. Stavridi E.S. Waterman J.L. Wieczorek A.M. Opella S.J. Halazonetis T.D. EMBO J. 1997; 16: 6230-6236Crossref PubMed Scopus (43) Google Scholar, 13Mateu M.G. Sanchez Del Pino M.M. Fersht A.R. Nat. Struct. Biol. 1999; 6: 191-198Crossref PubMed Scopus (100) Google Scholar, 14Noolandi J. Davison T.S. Volkel A.R. Nie X. Kay C. Arrowsmith C.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9955-9960Crossref PubMed Scopus (15) Google Scholar, 15Davison T.S. Nie X., Ma, W. Lin Y. Kay C. Benchimol S. Arrowsmith C.H. J. Mol. Biol. 2001; 307: 605-617Crossref PubMed Scopus (60) Google Scholar). To date, knowledge relating to the contribution of charged residues at this interface to the nature and stability of the tetramer through ion pair formation remains minimal. In comparison, the impact of ion pairs on the oligomeric state and stability of other self-assembling peptide domains such as coiled-coil sequences is well documented. Naturally occurring and engineered coiled-coil domains have been shown to form homodimers as well as heterodimers (16Graddis T.J. Myszka D.G. Chaiken I.M. Biochemistry. 1993; 32: 12664-12671Crossref PubMed Scopus (147) Google Scholar, 17Lavigne P. Kondejewski L.H. Houston M.E., Jr. Sonnichsen F.D. Lix B. Skyes B.D. Hodges R.S. Kay C.M. J. Mol. Biol. 1995; 254: 505-520Crossref PubMed Scopus (98) Google Scholar, 18Lumb K.J. Kim P.S. Biochemistry. 1995; 34: 8642-8648Crossref PubMed Scopus (300) Google Scholar, 19O'Shea E.K. Rutkowski R. Kim P.S. Cell. 1992; 68: 699-708Abstract Full Text PDF PubMed Scopus (370) Google Scholar) and heterotetramers (20Fairman R. Chao H.G. Lavoie T.B. Villafranca J.J. Matsueda G.R. Novotny J. Biochemistry. 1996; 35: 2824-2829Crossref PubMed Scopus (82) Google Scholar). Protein complexes such as the Fos-Jun heterodimer (19O'Shea E.K. Rutkowski R. Kim P.S. Cell. 1992; 68: 699-708Abstract Full Text PDF PubMed Scopus (370) Google Scholar), for example, occur as a result of charged groups in their coiled-coil regions, which promote hetero-oligomerization through the destabilization of homotypic interactions.An examination of the crystal structure (6Jeffrey P.D. Gorina S. Pavletich N.P. Science. 1995; 267: 1498-1502Crossref PubMed Scopus (435) Google Scholar) of the human p53 tetramerization domain reveals the presence of one arginine (Arg342), one lysine (Lys351), and four glutamates (Glu339, Glu343, Glu346, and Glu349) within the boundaries of the dimer-dimer interface (residues 338–351). Of these residues, the only pairs of complementary charged side chains proximal enough to form an intermonomer salt bridge involve Lys351 with Glu343 and/or Glu346 (see Fig. 1 b). The side chain oxygen of Glu343 on one monomer was found to be located 2.58 Å from the nitrogen side chain of Lys351on another monomer. NMR structures of p53tet (7Lee W. Harvey T.S. Yin Y. Yau P. Litchfield D. Arrowsmith C.H. Nat. Struct. Biol. 1994; 1: 877-890Crossref PubMed Scopus (234) Google Scholar, 21Clore G.M. Ernst J. Clubb R. Omichinski J.G. Kennedy W.M. Sakaguchi K. Appella E. Gronenborn A.M. Nat. Struct. Biol. 1995; 2: 321-333Crossref PubMed Scopus (188) Google Scholar) in solution have revealed that Glu346 is apparently closer than Glu343 to Lys351, although these ionic residues are farther apart in these structures than in the crystal structure. Finally, alignment of the tetramerization domain sequences of p53 fromXenopus laevis (22Soussi T. Caron de Fromentel C. Mechali M. May P. Kress M. Oncogene. 1987; 1: 71-78PubMed Google Scholar) and rainbow trout (23Caron de Fromentel C. Pakdel F. Chapus A. Baney C. May P. Soussi T. Gene (Amst.). 1992; 112: 241-245Crossref PubMed Scopus (69) Google Scholar) as well as of human p73 and p63 (24Kaghad M. Bonnet H. Yang A. Creancier L. Biscan J.C. Valent A. Minty A. Chalon P. Lelias J.M. Dumont X. Ferrara P. McKeon F. Caput D. Cell. 1997; 90: 809-819Abstract Full Text Full Text PDF PubMed Scopus (1533) Google Scholar, 25Davison T.S. Vagner C. Kaghad M. Ayed A. Caput D. Arrowsmith C.H. J. Biol. Chem. 1999; 274: 18709-18714Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 26Yang A. Kaghad M. Wang Y. Gillett E. Fleming M.D. Dotsch V. Andrews N.C. Caput D. McKeon F. Mol. Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1830) Google Scholar) (see Fig. 1 c) indicates that the naturally occurring E343K mutation in their tetramerization domains is always coupled with a corresponding loss of the positively charged lysine residue at position 351. Taken together, these findings suggest that the presence of salt bridges involving Lys351 with Glu343 and/or Glu346would contribute four or more ionic interactions within the context of the p53 tetramer interface, favoring the stabilization and self-association of primary dimers. To test this hypothesis, we engineered variants of the p53tet domain harboring charge-reversal mutations at positions 343, 346, and 351 (see Fig. 1 c). Mutants p53tet-E343K, p53tet-E346K, and p53tet-E343K/E346K will result in dimer-dimer interfaces exhibiting a preponderance of positively charged side chains, whereas mutant p53tet-K351E will produce an interface enriched for negatively charged residues, both scenarios leading to electrostatic interactions unfavorable for tetramer formation. Conversely, the negative effects of introducing like charges at the dimer-dimer interface should be nullified in the case of heterotetramers composed of dimers of p53tet-K351E paired with p53tet-E343K, p53tet-E346K, or p53tet-E343K/E346K. The stability and oligomeric state of these p53tet constructs were analyzed in a series of biophysical experiments to resolve the role of such salt bridges at the dimer-dimer interface.DISCUSSIONThe tetramerization domain of human p53 is an important part of this key tumor suppressor protein. Analysis of the dimer-dimer interface of the human p53 tetramerization domain suggests that ion pair interactions between Glu343, Glu346, and Lys351 may contribute significantly to the stability of the tetramer. This hypothesis was further supported by the fact that the tetramerization domain sequences of p53 in other organisms as well as of human p63 and p73 (Fig. 1 c) display the naturally occurring E343K mutation. This mutation is always coupled with a corresponding loss of the positively charged lysine residue at position 351. This hypothesis was tested by designing and analyzing variants of the tetramerization domain of human p53, viz. p53tet-E343K, p53tet-E346K, p53tet-E343K/E346K, and p53tet-K351E, harboring charge-reversal mutations at ionic residues.In the first part of this study, the oligomeric state of these p53tet mutants was evaluated (Fig. 3 and Table I) to determine whether these mutations, as is the case with many mutations of hydrophobic residues in the dimer-dimer interface (10Mateu M.G. Fersht A.R. EMBO J. 1998; 17: 2748-2758Crossref PubMed Scopus (126) Google Scholar, 12McCoy M. Stavridi E.S. Waterman J.L. Wieczorek A.M. Opella S.J. Halazonetis T.D. EMBO J. 1997; 16: 6230-6236Crossref PubMed Scopus (43) Google Scholar, 15Davison T.S. Nie X., Ma, W. Lin Y. Kay C. Benchimol S. Arrowsmith C.H. J. Mol. Biol. 2001; 307: 605-617Crossref PubMed Scopus (60) Google Scholar), change the oligomerization specificity of p53tet from a tetramer to a dimer. Indeed, it was revealed that p53tet-K351E is a dimer in solution, demonstrating that a single mutation to a charged residue is sufficient to produce dimeric p53. This finding confirms our hypothesis that the introduction of a charge-reversal mutation (Lys to Glu) at position 351 of the p53tet domain introduces non-constructive charge repulsions at the dimer-dimer interface.In the next part of this study, the stability of the resulting p53tet mutants was evaluated. It has been established that the oligomeric state and folding pattern of the p53 tetramerization domain are tightly linked features of this protein scaffold, with the monomeric form being essentially unfolded (10Mateu M.G. Fersht A.R. EMBO J. 1998; 17: 2748-2758Crossref PubMed Scopus (126) Google Scholar, 13Mateu M.G. Sanchez Del Pino M.M. Fersht A.R. Nat. Struct. Biol. 1999; 6: 191-198Crossref PubMed Scopus (100) Google Scholar, 32Johnson C.R. Morin P.E. Arrowsmith C.H. Freire E. Biochemistry. 1995; 34: 5309-5316Crossref PubMed Scopus (113) Google Scholar). Thus, thermal unfolding patterns as measured by CD represent an indicator of the tendency of the p53tet domain to oligomerize. As expected for a tetrameric protein, theT m of p53tet is dependent on protein concentration (10Mateu M.G. Fersht A.R. EMBO J. 1998; 17: 2748-2758Crossref PubMed Scopus (126) Google Scholar, 32Johnson C.R. Morin P.E. Arrowsmith C.H. Freire E. Biochemistry. 1995; 34: 5309-5316Crossref PubMed Scopus (113) Google Scholar). A p53 monomer concentration of 10 μm was thus selected for p53tet-WT to be fully unfolded at 98 °C, allowing us to compare T m values between p53tet-WT and its variants. The T m of p53tet is also dependent on the length of the protein or peptide used (10Mateu M.G. Fersht A.R. EMBO J. 1998; 17: 2748-2758Crossref PubMed Scopus (126) Google Scholar). However, the observedT m of 68 °C for the 72-amino acid-long p53tet-WT construct used in this study is comparable to published values for related p53 constructs analyzed under these conditions (9DiGiammarino E.L. Lee A.S. Cadwell C. Zhang W. Bothner B. Ribeiro R.C. Zambetti G. Kriwacki R.W. Nat. Struct. Biol. 2002; 9: 12-16Crossref PubMed Scopus (213) Google Scholar, 10Mateu M.G. Fersht A.R. EMBO J. 1998; 17: 2748-2758Crossref PubMed Scopus (126) Google Scholar, 32Johnson C.R. Morin P.E. Arrowsmith C.H. Freire E. Biochemistry. 1995; 34: 5309-5316Crossref PubMed Scopus (113) Google Scholar). The thermal unfolding results shown in Fig. 4 revealed that p53tet-E346K was less stable than p53tet-E343K, suggesting that Glu346is involved in a more pronounced stabilizing interaction at the dimer-dimer interface. Glu346 (rather than Glu343) may thus more strongly interact with Lys351, in contrast with predictions arising from the crystal structure (6Jeffrey P.D. Gorina S. Pavletich N.P. Science. 1995; 267: 1498-1502Crossref PubMed Scopus (435) Google Scholar). CD thermal unfolding studies showed that p53tet-K351E, in addition to being a dimer, was also very unstable, suggesting that the charge at position 351 is an important determinant of the stability of the p53 tetramer.Subsequent experiments were undertaken to determine the potential of these p53tet mutants to form heterotetramers. SEC (Fig. 3) and analytical ultracentrifugation (Table I) data both indicated that either the E343K or E346K mutation was sufficient to produce a species that specifically formed heterotetramers with p53tet-K351E. However, CD data (Fig. 4 and Table II) suggested that when both E343K and E346K mutations were included, the resulting heterotetramer with p53tet-K351E was much more stable relative to the two individual components. The specificity of the heterotetramer between p53tet-E343K/E346K and p53tet-K351E was also confirmed by metal affinity experiments (Fig. 5), which strikingly depicted the necessity of both Glu-to-Lys mutations in determining the specificity of the heterotetramer. It was also found that these two mutants specifically associated with each other, and not with wild-type human p53tet. This interesting finding suggests that human p53 mutants containing such mutations would indeed not have a 'dominant-negative' effect on cellular transformation because their intracellular expression would not directly compete or exchange with existing cellular pools of wild-type human p53 (33Chene P. Bechter E. J. Mol. Biol. 1999; 286: 1269-1274Crossref PubMed Scopus (27) Google Scholar, 34Shaulian E. Zauberman A. Ginsberg D. Oren M. Mol. Cell. Biol. 1992; 12: 5581-5592Crossref PubMed Scopus (322) Google Scholar). This study demonstrates for the first time the important contribution of ionic interactions involving Glu343, Glu346, and Lys351 to the stability of the dimer-dimer interface of the human p53tet domain. Human p53 plays an important role in tumor suppression (1Lane D.P. Nature. 1992; 358: 15-16Crossref PubMed Scopus (4437) Google Scholar, 2Arrowsmith C.H. Morin P. Oncogene. 1996; 12: 1379-1385PubMed Google Scholar, 3Bargonetti J. Manfredi J.J. Curr. Opin. Oncol. 2002; 14: 86-91Crossref PubMed Scopus (311) Google Scholar, 4Somasundaram K. Front. Biosci. 2000; 5: D424-D437Crossref PubMed Google Scholar). It is a modular protein consisting of discrete functional domains, which can be expressed and studied in isolation. In particular, residues 325–355 of human p53 (p53tet) spontaneously form a tetramer in solution (see Fig. 1 a) (5Chene P. Oncogene. 2001; 20: 2611-2617Crossref PubMed Scopus (202) Google Scholar, 6Jeffrey P.D. Gorina S. Pavletich N.P. Science. 1995; 267: 1498-1502Crossref PubMed Scopus (435) Google Scholar, 7Lee W. Harvey T.S. Yin Y. Yau P. Litchfield D. Arrowsmith C.H. Nat. Struct. Biol. 1994; 1: 877-890Crossref PubMed Scopus (234) Google Scholar). Each monomer within the context of the p53tet domain adopts an identical structure,viz. a short N-terminal β-strand (residues 326–333) followed by a turn and a C-terminal α-helical domain (residues 335–354). Two monomers associate in an antiparallel fashion through contacts between β-sheet strands as well as hydrophobic interactions involving α-helical residues to form a "primary dimer" (6Jeffrey P.D. Gorina S. Pavletich N.P. Science. 1995; 267: 1498-1502Crossref PubMed Scopus (435) Google Scholar, 7Lee W. Harvey T.S. Yin Y. Yau P. Litchfield D. Arrowsmith C.H. Nat. Struct. Biol. 1994; 1: 877-890Crossref PubMed Scopus (234) Google Scholar). One significant salt bridge in the p53tet region occurs between Arg337 of one subunit and Asp352 of its adjacent subunit (side chain oxygen–nitrogen distance of 2.72 Å) (6Jeffrey P.D. Gorina S. Pavletich N.P. Science. 1995; 267: 1498-1502Crossref PubMed Scopus (435) Google Scholar), stabilizing the structure of the primary dimer (8Davison T.S. Yin P. Nie E. Kay C. Arrowsmith C.H. Oncogene. 1998; 17: 651-656Crossref PubMed Scopus (94) Google Scholar, 9DiGiammarino E.L. Lee A.S. Cadwell C. Zhang W. Bothner B. Ribeiro R.C. Zambetti G. Kriwacki R.W. Nat. Struct. Biol. 2002; 9: 12-16Crossref PubMed Scopus (213) Google Scholar). Two primary dimers then self-associate through an interface derived from residues located in their α-helical domains to form a "dimer of dimers," referred to as a p53 tetramer. Mutations of amino acids at this interface have highlighted the importance of hydrophobic residues leading to the formation of the tetramer as well as stable p53 dimers (10Mateu M.G. Fersht A.R. EMBO J. 1998; 17: 2748-2758Crossref PubMed Scopus (126) Google Scholar, 11Mateu M.G. Fersht A.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3595-3599Crossref PubMed Scopus (59) Google Scholar, 12McCoy M. Stavridi E.S. Waterman J.L. Wieczorek A.M. Opella S.J. Halazonetis T.D. EMBO J. 1997; 16: 6230-6236Crossref PubMed Scopus (43) Google Scholar, 13Mateu M.G. Sanchez Del Pino M.M. Fersht A.R. Nat. Struct. Biol. 1999; 6: 191-198Crossref PubMed Scopus (100) Google Scholar, 14Noolandi J. Davison T.S. Volkel A.R. Nie X. Kay C. Arrowsmith C.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9955-9960Crossref PubMed Scopus (15) Google Scholar, 15Davison T.S. Nie X., Ma, W. Lin Y. Kay C. Benchimol S. Arrowsmith C.H. J. Mol. Biol. 2001; 307: 605-617Crossref PubMed Scopus (60) Google Scholar). To date, knowledge relating to the contribution of charged residues at this interface to the nature and stability of the tetramer through ion pair formation remains minimal. In comparison, the impact of ion pairs on the oligomeric state and stability of other self-assembling peptide domains such as coiled-coil sequences is well documented. Naturally occurring and engineered coiled-coil domains have been shown to form homodimers as well as heterodimers (16Graddis T.J. Myszka D.G. Chaiken I.M. Biochemistry. 1993; 32: 12664-12671Crossref PubMed Scopus (147) Google Scholar, 17Lavigne P. Kondejewski L.H. Houston M.E., Jr. Sonnichsen F.D. Lix B. Skyes B.D. Hodges R.S. Kay C.M. J. Mol. Biol. 1995; 254: 505-520Crossref PubMed Scopus (98) Google Scholar, 18Lumb K.J. Kim P.S. Biochemistry. 1995; 34: 8642-8648Crossref PubMed Scopus (300) Google Scholar, 19O'Shea E.K. Rutkowski R. Kim P.S. Cell. 1992; 68: 699-708Abstract Full Text PDF PubMed Scopus (370) Google Scholar) and heterotetramers (20Fairman R. Chao H.G. Lavoie T.B. Villafranca J.J. Matsueda G.R. Novotny J. Biochemistry. 1996; 35: 2824-2829Crossref PubMed Scopus (82) Google Scholar). Protein complexes such as the Fos-Jun heterodimer (19O'Shea E.K. Rutkowski R. Kim P.S. Cell. 1992; 68: 699-708Abstract Full Text PDF PubMed Scopus (370) Google Scholar), for example, occur as a result of charged groups in their coiled-coil regions, which promote hetero-oligomerization through the destabilization of homotypic interactions. An examination of the crystal structure (6Jeffrey P.D. Gorina S. Pavletich N.P. Science. 1995; 267: 1498-1502Crossref PubMed Scopus (435) Google Scholar) of the human p53 tetramerization domain reveals the presence of one arginine (Arg342), one lysine (Lys351), and four glutamates (Glu339, Glu343, Glu346, and Glu349) within the boundaries of the dimer-dimer interface (residues 338–351). Of these residues, the only pairs of complementary charged side chains proximal enough to form an intermonomer salt bridge involve Lys351 with Glu343 and/or Glu346 (see Fig. 1 b). The side chain oxygen of Glu343 on one monomer was found to be located 2.58 Å from the nitrogen side chain of Lys351on another monomer. NMR structures of p53tet (7Lee W. Harvey T.S. Yin Y. Yau P. Litchfield D. Arrowsmith C.H. Nat. Struct. Biol. 1994; 1: 877-890Crossref PubMed Scopus (234) Google Scholar, 21Clore G.M. Ernst J. Clubb R. Omichinski J.G. Kennedy W.M. Sakaguchi K. Appella E. Gronenborn A.M. Nat. Struct. Biol. 1995; 2: 321-333Crossref PubMed Scopus (188) Google Scholar) in solution have revealed that Glu346 is apparently closer than Glu343 to Lys351, although these ionic residues are farther apart in these structures than in the crystal structure. Finally, alignment of the tetramerization domain sequences of p53 fromXenopus laevis (22Soussi T. Caron de Fromentel C. Mechali M. May P. Kress M. Oncogene. 1987; 1: 71-78PubMed Google Scholar) and rainbow trout (23Caron de Fromentel C. Pakdel F. Chapus A. Baney C. May P. Soussi T. Gene (Amst.). 1992; 112: 241-245Crossref PubMed Scopus (69) Google Scholar) as well as of human p73 and p63 (24Kaghad M. Bonnet H. Yang A. Creancier L. Biscan J.C. Valent A. Minty A. Chalon P. Lelias J.M. Dumont X. Ferrara P. McKeon F. Caput D. Cell. 1997; 90: 809-819Abstract Full Text Full Text PDF PubMed Scopus (1533) Google Scholar, 25Davison T.S. Vagner C. Kaghad M. Ayed A. Caput D. Arrowsmith C.H. J. Biol. Chem. 1999; 274: 18709-18714Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 26Yang A. Kaghad M. Wang Y. Gillett E. Fleming M.D. Dotsch V. Andrews N.C. Caput D. McKeon F. Mol. Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1830) Google Scholar) (see Fig. 1 c) indicates that the naturally occurring E343K mutation in their tetramerization domains is always coupled with a corresponding loss of the positively charged lysine residue at position 351. Taken together, these findings suggest that the presence of salt bridges involving Lys351 with Glu343 and/or Glu346would contribute four or more ionic interactions within the context of the p53 tetramer interface, favoring the stabilization and self-association of primary dimers. To test this hypothesis, we engineered variants of the p53tet domain harboring charge-reversal mutations at positions 343, 346, and 351 (see Fig. 1 c). Mutants p53tet-E343K, p53tet-E346K, and p53tet-E343K/E346K will result in dimer-dimer interfaces exhibiting a preponderance of positively charged side chains, whereas mutant p53tet-K351E will produce an interface enriched for negatively charged residues, both scenarios leading to electrostatic interactions unfavorable for tetramer formation. Conversely, the negative effects of introducing like charges at the dimer-dimer interface should be nullified in the case of heterotetramers composed of dimers of p53tet-K351E paired with p53tet-E343K, p53tet-E346K, or p53tet-E343K/E346K. The stability and oligomeric state of these p53tet constructs were analyzed in a series of biophysical experiments to resolve the role of such salt bridges at the dimer-dimer interface. DISCUSSIONThe tetramerization domain of human p53 is an important part of this key tumor suppressor protein. Analysis of the dimer-dimer interface of the human p53 tetramerization domain suggests that ion pair interactions between Glu343, Glu346, and Lys351 may contribute significantly to the stability of the tetramer. This hypothesis was further supported by the fact that the tetramerization domain sequences of p53 in other organisms as well as of human p63 and p73 (Fig. 1 c) display the naturally occurring E343K mutation. This mutation is always coupled with a corresponding loss of the positively charged lysine residue at position 351. This hypothesis was tested by designing and analyzing variants of the tetramerization domain of human p53, viz. p53tet-E343K, p53tet-E346K, p53tet-E343K/E346K, and p53tet-K351E, harboring charge-reversal mutations at ionic residues.In the first part of this study, the oligomeric state of these p53tet mutants was evaluated (Fig. 3 and Table I) to determine whether these mutations, as is the case with many mutations of hydrophobic residues in the dimer-dimer interface (10Mateu M.G. Fersht A.R. EMBO J. 1998; 17: 2748-2758Crossref PubMed Scopus (126) Google Scholar, 12McCoy M. Stavridi E.S. Waterman J.L. Wieczorek A.M. Opella S.J. Halazonetis T.D. EMBO J. 1997; 16: 6230-6236Crossref PubMed Scopus (43) Google Scholar, 15Davison T.S. Nie X., Ma, W. Lin Y. Kay C. Benchimol S. Arrowsmith C.H. J. Mol. Biol. 2001; 307: 605-617Crossref PubMed Scopus (60) Google Scholar), change the oligomerization specificity of p53tet from a tetramer to a dimer. Indeed, it was revealed that p53tet-K351E is a dimer in solution, demonstrating that a single mutation to a charged residue is sufficient to produce dimeric p53. This finding confirms our hypothesis that the introduction of a charge-reversal mutation (Lys to Glu) at position 351 of the p53tet domain introduces non-constructive charge repulsions at the dimer-dimer interface.In the next part of this study, the stability of the resulting p53tet mutants was evaluated. It has been established that the oligomeric state and folding pattern of the p53 tetramerization domain are tightly linked features of this protein scaffold, with the monomeric form being essentially unfolded (10Mateu M.G. Fersht A.R. EMBO J. 1998; 17: 2748-2758Crossref PubMed Scopus (126) Google Scholar, 13Mateu M.G. Sanchez Del Pino M.M. Fersht A.R. Nat. Struct. Biol. 1999; 6: 191-198Crossref PubMed Scopus (100) Google Scholar, 32Johnson C.R. Morin P.E. Arrowsmith C.H. Freire E. Biochemistry. 1995; 34: 5309-5316Crossref PubMed Scopus (113) Google Scholar). Thus, thermal unfolding patterns as measured by CD represent an indicator of the tendency of the p53tet domain to oligomerize. As expected for a tetrameric protein, theT m of p53tet is dependent on protein concentration (10Mateu M.G. Fersht A.R. EMBO J. 1998; 17: 2748-2758Crossref PubMed Scopus (126) Google Scholar, 32Johnson C.R. Morin P.E. Arrowsmith C.H. Freire E. Biochemistry. 1995; 34: 5309-5316Crossref PubMed Scopus (113) Google Scholar). A p53 monomer concentration of 10 μm was thus selected for p53tet-WT to be fully unfolded at 98 °C, allowing us to compare T m values between p53tet-WT and its variants. The T m of p53tet is also dependent on the length of the protein or peptide used (10Mateu M.G. Fersht A.R. EMBO J. 1998; 17: 2748-2758Crossref PubMed Scopus (126) Google Scholar). However, the observedT m of 68 °C for the 72-amino acid-long p53tet-WT construct used in this study is comparable to published values for related p53 constructs analyzed under these conditions (9DiGiammarino E.L. Lee A.S. Cadwell C. Zhang W. Bothner B. Ribeiro R.C. Zambetti G. Kriwacki R.W. Nat. Struct. Biol. 2002; 9: 12-16Crossref PubMed Scopus (213) Google Scholar, 10Mateu M.G. Fersht A.R. EMBO J. 1998; 17: 2748-2758Crossref PubMed Scopus (126) Google Scholar, 32Johnson C.R. Morin P.E. Arrowsmith C.H. Freire E. Biochemistry. 1995; 34: 5309-5316Crossref PubMed Scopus (113) Google Scholar). The thermal unfolding results shown in Fig. 4 revealed that p53tet-E346K was less stable than p53tet-E343K, suggesting that Glu346is involved in a more pronounced stabilizing interaction at the dimer-dimer interface. Glu346 (rather than Glu343) may thus more strongly interact with Lys351, in contrast with predictions arising from the crystal structure (6Jeffrey P.D. Gorina S. Pavletich N.P. Science. 1995; 267: 1498-1502Crossref PubMed Scopus (435) Google Scholar). CD thermal unfolding studies showed that p53tet-K351E, in addition to being a dimer, was also very unstable, suggesting that the charge at position 351 is an important determinant of the stability of the p53 tetramer.Subsequent experiments were undertaken to determine the potential of these p53tet mutants to form heterotetramers. SEC (Fig. 3) and analytical ultracentrifugation (Table I) data both indicated that either the E343K or E346K mutation was sufficient to produce a species that specifically formed heterotetramers with p53tet-K351E. However, CD data (Fig. 4 and Table II) suggested that when both E343K and E346K mutations were included, the resulting heterotetramer with p53tet-K351E was much more stable relative to the two individual components. The specificity of the heterotetramer between p53tet-E343K/E346K and p53tet-K351E was also confirmed by metal affinity experiments (Fig. 5), which strikingly depicted the necessity of both Glu-to-Lys mutations in determining the specificity of the heterotetramer. It was also found that these two mutants specifically associated with each other, and not with wild-type human p53tet. This interesting finding suggests that human p53 mutants containing such mutations would indeed not have a 'dominant-negative' effect on cellular transformation because their intracellular expression would not directly compete or exchange with existing cellular pools of wild-type human p53 (33Chene P. Bechter E. J. Mol. Biol. 1999; 286: 1269-1274Crossref PubMed Scopus (27) Google Scholar, 34Shaulian E. Zauberman A. Ginsberg D. Oren M. Mol. Cell. Biol. 1992; 12: 5581-5592Crossref PubMed Scopus (322) Google Scholar). This study demonstrates for the first time the important contribution of ionic interactions involving Glu343, Glu346, and Lys351 to the stability of the dimer-dimer interface of the human p53tet domain. The tetramerization domain of human p53 is an important part of this key tumor suppressor protein. Analysis of the dimer-dimer interface of the human p53 tetramerization domain suggests that ion pair interactions between Glu343, Glu346, and Lys351 may contribute significantly to the stability of the tetramer. This hypothesis was further supported by the fact that the tetramerization domain sequences of p53 in other organisms as well as of human p63 and p73 (Fig. 1 c) display the naturally occurring E343K mutation. This mutation is always coupled with a corresponding loss of the positively charged lysine residue at position 351. This hypothesis was tested by designing and analyzing variants of the tetramerization domain of human p53, viz. p53tet-E343K, p53tet-E346K, p53tet-E343K/E346K, and p53tet-K351E, harboring charge-reversal mutations at ionic residues. In the first part of this study, the oligomeric state of these p53tet mutants was evaluated (Fig. 3 and Table I) to determine whether these mutations, as is the case with many mutations of hydrophobic residues in the dimer-dimer interface (10Mateu M.G. Fersht A.R. EMBO J. 1998; 17: 2748-2758Crossref PubMed Scopus (126) Google Scholar, 12McCoy M. Stavridi E.S. Waterman J.L. Wieczorek A.M. Opella S.J. Halazonetis T.D. EMBO J. 1997; 16: 6230-6236Crossref PubMed Scopus (43) Google Scholar, 15Davison T.S. Nie X., Ma, W. Lin Y. Kay C. Benchimol S. Arrowsmith C.H. J. Mol. Biol. 2001; 307: 605-617Crossref PubMed Scopus (60) Google Scholar), change the oligomerization specificity of p53tet from a tetramer to a dimer. Indeed, it was revealed that p53tet-K351E is a dimer in solution, demonstrating that a single mutation to a charged residue is sufficient to produce dimeric p53. This finding confirms our hypothesis that the introduction of a charge-reversal mutation (Lys to Glu) at position 351 of the p53tet domain introduces non-constructive charge repulsions at the dimer-dimer interface. In the next part of this study, the stability of the resulting p53tet mutants was evaluated. It has been established that the oligomeric state and folding pattern of the p53 tetramerization domain are tightly linked features of this protein scaffold, with the monomeric form being essentially unfolded (10Mateu M.G. Fersht A.R. EMBO J. 1998; 17: 2748-2758Crossref PubMed Scopus (126) Google Scholar, 13Mateu M.G. Sanchez Del Pino M.M. Fersht A.R. Nat. Struct. Biol. 1999; 6: 191-198Crossref PubMed Scopus (100) Google Scholar, 32Johnson C.R. Morin P.E. Arrowsmith C.H. Freire E. Biochemistry. 1995; 34: 5309-5316Crossref PubMed Scopus (113) Google Scholar). Thus, thermal unfolding patterns as measured by CD represent an indicator of the tendency of the p53tet domain to oligomerize. As expected for a tetrameric protein, theT m of p53tet is dependent on protein concentration (10Mateu M.G. Fersht A.R. EMBO J. 1998; 17: 2748-2758Crossref PubMed Scopus (126) Google Scholar, 32Johnson C.R. Morin P.E. Arrowsmith C.H. Freire E. Biochemistry. 1995; 34: 5309-5316Crossref PubMed Scopus (113) Google Scholar). A p53 monomer concentration of 10 μm was thus selected for p53tet-WT to be fully unfolded at 98 °C, allowing us to compare T m values between p53tet-WT and its variants. The T m of p53tet is also dependent on the length of the protein or peptide used (10Mateu M.G. Fersht A.R. EMBO J. 1998; 17: 2748-2758Crossref PubMed Scopus (126) Google Scholar). However, the observedT m of 68 °C for the 72-amino acid-long p53tet-WT construct used in this study is comparable to published values for related p53 constructs analyzed under these conditions (9DiGiammarino E.L. Lee A.S. Cadwell C. Zhang W. Bothner B. Ribeiro R.C. Zambetti G. Kriwacki R.W. Nat. Struct. Biol. 2002; 9: 12-16Crossref PubMed Scopus (213) Google Scholar, 10Mateu M.G. Fersht A.R. EMBO J. 1998; 17: 2748-2758Crossref PubMed Scopus (126) Google Scholar, 32Johnson C.R. Morin P.E. Arrowsmith C.H. Freire E. Biochemistry. 1995; 34: 5309-5316Crossref PubMed Scopus (113) Google Scholar). The thermal unfolding results shown in Fig. 4 revealed that p53tet-E346K was less stable than p53tet-E343K, suggesting that Glu346is involved in a more pronounced stabilizing interaction at the dimer-dimer interface. Glu346 (rather than Glu343) may thus more strongly interact with Lys351, in contrast with predictions arising from the crystal structure (6Jeffrey P.D. Gorina S. Pavletich N.P. Science. 1995; 267: 1498-1502Crossref PubMed Scopus (435) Google Scholar). CD thermal unfolding studies showed that p53tet-K351E, in addition to being a dimer, was also very unstable, suggesting that the charge at position 351 is an important determinant of the stability of the p53 tetramer. Subsequent experiments were undertaken to determine the potential of these p53tet mutants to form heterotetramers. SEC (Fig. 3) and analytical ultracentrifugation (Table I) data both indicated that either the E343K or E346K mutation was sufficient to produce a species that specifically formed heterotetramers with p53tet-K351E. However, CD data (Fig. 4 and Table II) suggested that when both E343K and E346K mutations were included, the resulting heterotetramer with p53tet-K351E was much more stable relative to the two individual components. The specificity of the heterotetramer between p53tet-E343K/E346K and p53tet-K351E was also confirmed by metal affinity experiments (Fig. 5), which strikingly depicted the necessity of both Glu-to-Lys mutations in determining the specificity of the heterotetramer. It was also found that these two mutants specifically associated with each other, and not with wild-type human p53tet. This interesting finding suggests that human p53 mutants containing such mutations would indeed not have a 'dominant-negative' effect on cellular transformation because their intracellular expression would not directly compete or exchange with existing cellular pools of wild-type human p53 (33Chene P. Bechter E. J. Mol. Biol. 1999; 286: 1269-1274Crossref PubMed Scopus (27) Google Scholar, 34Shaulian E. Zauberman A. Ginsberg D. Oren M. Mol. Cell. Biol. 1992; 12: 5581-5592Crossref PubMed Scopus (322) Google Scholar). This study demonstrates for the first time the important contribution of ionic interactions involving Glu343, Glu346, and Lys351 to the stability of the dimer-dimer interface of the human p53tet domain. We thank Dr. Cheryl Arrowsmith for providing the pET-15b-p53-(310–360) plasmid and the pET-15b-p53-(310–360)-M340Q/L344R mutant plasmid used in this study and for critical review of this manuscript. Sandy Go and Dr. Avi Chakrabartty are acknowledged for performing the analytical ultracentrifugation experiments.
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