A Stable Human p53 Heterotetramer Based on Constructive Charge Interactions within the Tetramerization Domain

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. 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; PubMed Scopus Google Scholar) in solution have that Glu346 is Glu343 to these ionic residues in these structures in the crystal of the tetramerization domain sequences of p53 C. M. P. M. Oncogene. 1: Google Scholar) and C. A. C. P. 1992; PubMed Scopus Google Scholar) as well as of human and M. A. A. A. P. X. P. D. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, T.S. C. M. A. D. Arrowsmith C.H. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, A. M. Y. E. D. Mol. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) (see Fig. 1 that the occurring in their tetramerization domains is with a of the charged lysine at these that the presence of salt involving Lys351 with Glu343 and/or contribute four ionic interactions within the context of the p53 tetramer the and of primary To this engineered of the p53tet domain mutations at positions 343, 346, and 351 (see Fig. 1 and p53tet-E343K/E346K result in dimer-dimer a of charged side whereas mutant p53tet-K351E an interface for charged residues, leading to interactions for tetramer the of at the dimer-dimer interface be in the of heterotetramers composed of dimers of p53tet-K351E with The stability and oligomeric state of these p53tet constructs were in a of to the role of such salt at the dimer-dimer tetramerization domain of human p53 is an important of this tumor of the dimer-dimer interface of the human p53 tetramerization domain suggests that ion pair interactions between Glu343, Glu346, and Lys351 contribute to the stability of the tetramer. This was by the that the tetramerization domain sequences of p53 in other as well as of human and 1 the occurring This is with a of the charged lysine at This was by and of the tetramerization domain of human and mutations at ionic the of this the oligomeric state of these p53tet mutants was and to these as is the with 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), the specificity of p53tet from a tetramer to a dimer. was that p53tet-K351E is a dimer in solution, that a to a charged is to This that the of a to at 351 of the p53tet domain at the dimer-dimer the of this the stability of the p53tet mutants was It been that the oligomeric state and of the p53 tetramerization domain of this protein with the form (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, Morin Arrowsmith C.H. E. Biochemistry. 1995; 34: PubMed Scopus Google Scholar). thermal as by an of the of the p53tet domain to for a of p53tet is on protein (10Mateu M.G. Fersht A.R. EMBO J. 1998; 17: 2748-2758Crossref PubMed Scopus (126) Google Scholar, Morin Arrowsmith C.H. E. Biochemistry. 1995; 34: PubMed Scopus Google Scholar). p53 monomer of was thus for to be at to between and its The of p53tet is also on the of the protein peptide (10Mateu M.G. Fersht A.R. EMBO J. 1998; 17: 2748-2758Crossref PubMed Scopus (126) Google Scholar). However, the of for the in this study is to for p53 constructs these 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, M.G. Fersht A.R. EMBO J. 1998; 17: 2748-2758Crossref PubMed Scopus (126) Google Scholar, Morin Arrowsmith C.H. E. Biochemistry. 1995; 34: PubMed Scopus Google Scholar). The thermal shown in Fig. that was stable that in a stabilizing at the dimer-dimer Glu346 thus with in with from the crystal structure (6Jeffrey P.D. Gorina S. Pavletich N.P. Science. 1995; 267: 1498-1502Crossref PubMed Scopus (435) Google Scholar). thermal that in to a was also that the at 351 is an important of the stability of the p53 were to the of these p53tet mutants to form and that either the was to a that heterotetramers with However, and that and mutations were the heterotetramer with p53tet-K351E was stable relative to the two The specificity of the heterotetramer between p53tet-E343K/E346K and p53tet-K351E was also by which the of mutations in the specificity of the heterotetramer. It was also found that these two mutants associated with each other, and not with wild-type human This suggests that human p53 mutants such mutations not have a on their not with of wild-type human p53 P. E. J. Mol. Biol. 1999; PubMed Scopus Google Scholar, E. A. D. M. Mol. Cell. Biol. 1992; 12: PubMed Scopus Google Scholar). This study demonstrates for the the important contribution of ionic interactions involving Glu343, Glu346, and Lys351 to the stability of the dimer-dimer interface of the human p53tet 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 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; PubMed Scopus Google Scholar) in solution have that Glu346 is Glu343 to these ionic residues in these structures in the crystal of the tetramerization domain sequences of p53 C. M. P. M. Oncogene. 1: Google Scholar) and C. A. C. P. 1992; PubMed Scopus Google Scholar) as well as of human and M. A. A. A. P. X. P. D. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, T.S. C. M. A. D. Arrowsmith C.H. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, A. M. Y. E. D. Mol. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) (see Fig. 1 that the occurring in their tetramerization domains is with a of the charged lysine at these that the presence of salt involving Lys351 with Glu343 and/or contribute four ionic interactions within the context of the p53 tetramer the and of primary To this engineered of the p53tet domain mutations at positions 343, 346, and 351 (see Fig. 1 and p53tet-E343K/E346K result in dimer-dimer a of charged side whereas mutant p53tet-K351E an interface for charged residues, leading to interactions for tetramer the of at the dimer-dimer interface be in the of heterotetramers composed of dimers of p53tet-K351E with The stability and oligomeric state of these p53tet constructs were in a of to the role of such salt at the dimer-dimer tetramerization domain of human p53 is an important of this tumor of the dimer-dimer interface of the human p53 tetramerization domain suggests that ion pair interactions between Glu343, Glu346, and Lys351 contribute to the stability of the tetramer. This was by the that the tetramerization domain sequences of p53 in other as well as of human and 1 the occurring This is with a of the charged lysine at This was by and of the tetramerization domain of human and mutations at ionic the of this the oligomeric state of these p53tet mutants was and to these as is the with 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), the specificity of p53tet from a tetramer to a dimer. was that p53tet-K351E is a dimer in solution, that a to a charged is to This that the of a to at 351 of the p53tet domain at the dimer-dimer the of this the stability of the p53tet mutants was It been that the oligomeric state and of the p53 tetramerization domain of this protein with the form (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, Morin Arrowsmith C.H. E. Biochemistry. 1995; 34: PubMed Scopus Google Scholar). thermal as by an of the of the p53tet domain to for a of p53tet is on protein (10Mateu M.G. Fersht A.R. EMBO J. 1998; 17: 2748-2758Crossref PubMed Scopus (126) Google Scholar, Morin Arrowsmith C.H. E. Biochemistry. 1995; 34: PubMed Scopus Google Scholar). p53 monomer of was thus for to be at to between and its The of p53tet is also on the of the protein peptide (10Mateu M.G. Fersht A.R. EMBO J. 1998; 17: 2748-2758Crossref PubMed Scopus (126) Google Scholar). However, the of for the in this study is to for p53 constructs these 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, M.G. Fersht A.R. EMBO J. 1998; 17: 2748-2758Crossref PubMed Scopus (126) Google Scholar, Morin Arrowsmith C.H. E. Biochemistry. 1995; 34: PubMed Scopus Google Scholar). The thermal shown in Fig. that was stable that in a stabilizing at the dimer-dimer Glu346 thus with in with from the crystal structure (6Jeffrey P.D. Gorina S. Pavletich N.P. Science. 1995; 267: 1498-1502Crossref PubMed Scopus (435) Google Scholar). thermal that in to a was also that the at 351 is an important of the stability of the p53 were to the of these p53tet mutants to form and that either the was to a that heterotetramers with However, and that and mutations were the heterotetramer with p53tet-K351E was stable relative to the two The specificity of the heterotetramer between p53tet-E343K/E346K and p53tet-K351E was also by which the of mutations in the specificity of the heterotetramer. It was also found that these two mutants associated with each other, and not with wild-type human This suggests that human p53 mutants such mutations not have a on their not with of wild-type human p53 P. E. J. Mol. Biol. 1999; PubMed Scopus Google Scholar, E. A. D. M. Mol. Cell. Biol. 1992; 12: PubMed Scopus Google Scholar). This study demonstrates for the the important contribution of ionic interactions involving Glu343, Glu346, and Lys351 to the stability of the dimer-dimer interface of the human p53tet The tetramerization domain of human p53 is an important of this tumor of the dimer-dimer interface of the human p53 tetramerization domain suggests that ion pair interactions between Glu343, Glu346, and Lys351 contribute to the stability of the tetramer. This was by the that the tetramerization domain sequences of p53 in other as well as of human and 1 the occurring This is with a of the charged lysine at This was by and of the tetramerization domain of human and mutations at ionic In the of this the oligomeric state of these p53tet mutants was and to these as is the with 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), the specificity of p53tet from a tetramer to a dimer. was that p53tet-K351E is a dimer in solution, that a to a charged is to This that the of a to at 351 of the p53tet domain at the dimer-dimer In the of this the stability of the p53tet mutants was It been that the oligomeric state and of the p53 tetramerization domain of this protein with the form (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, Morin Arrowsmith C.H. E. Biochemistry. 1995; 34: PubMed Scopus Google Scholar). thermal as by an of the of the p53tet domain to for a of p53tet is on protein (10Mateu M.G. Fersht A.R. EMBO J. 1998; 17: 2748-2758Crossref PubMed Scopus (126) Google Scholar, Morin Arrowsmith C.H. E. Biochemistry. 1995; 34: PubMed Scopus Google Scholar). p53 monomer of was thus for to be at to between and its The of p53tet is also on the of the protein peptide (10Mateu M.G. Fersht A.R. EMBO J. 1998; 17: 2748-2758Crossref PubMed Scopus (126) Google Scholar). However, the of for the in this study is to for p53 constructs these 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, M.G. Fersht A.R. EMBO J. 1998; 17: 2748-2758Crossref PubMed Scopus (126) Google Scholar, Morin Arrowsmith C.H. E. Biochemistry. 1995; 34: PubMed Scopus Google Scholar). The thermal shown in Fig. that was stable that in a stabilizing at the dimer-dimer Glu346 thus with in with from the crystal structure (6Jeffrey P.D. Gorina S. Pavletich N.P. 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This study demonstrates for the the important contribution of ionic interactions involving Glu343, Glu346, and Lys351 to the stability of the dimer-dimer interface of the human p53tet Arrowsmith for the and the mutant in this study and for of this and for the

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