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How loops, β sheets, and α helices help us to understand p53
Cell, Vol. 76, 543-546,
August
26, 1994, Copyright
0 1994 by Cell Press
How Loops, p Sheets, and a Helices Help Us to Understand ~53 Carol Prives Department of Biological Sciences Columbia University New York, New York 10027
~53 plays a critical role in transducing a signal from damaged DNA to genes that control the cell cycle and apoptosis. Intrinsic to its function in this pathway is the ability of ~53, a tetrameric phosphoprotein, to bind sequence specifically to DNA. This property allows ~53 to serve as a transcriptional activator. Several ~53 target genes have been identified that contain a ~53 response element that conforms to the consensus sequence (5-R R R C AIT TIA G Y Y Y-33*. Tumor-derived ~53 mutants almost invariably display abnormal DNA binding and transcriptional activation properties. These and other relevant facts have been discussed in recent reviews (Levine, 1993; Prives and Manfredi, 1993). p53 Mutations in Cancer Experimental manipulation of the ~53 gene and protein has been useful for unveiling its functions and domains. However, there has been an equally powerful and revealing set of information about ~53 that has not been derived by experimentation-that resulting from sequencing the tumor-derived ~53 genes of nearly 2000 cancer patients (Figure 1; Lin et al., 1994; Hollstein et al., 1994). When aligned with the sequence of ~53 and compared with what is now known about its functions and properties, these
pSSbinding trnnseriplionalXlivPlion Ad Elb
SV4S T ml&en bindiq
TBP bind&
I
r
sequence-specific
mutants provide an extraordinary view of the ~53 protein. First, thevast majority of the mutationsare clustered within the central portion of the protein, where 4 of the 5 regions that are highly conserved among all ~53 genes sequenced to date are located. Second, although mutation of the great majority (but not all) of the amino acids within the center of p53 has occurred in tumors, there are among these a number of “hot spots” that occur with unusually high frequency. This spectrum of ~53 mutations tells us that central to the role of ~53 in preventing cancer is the very center of p53 itself. The Domains of p53 Reverse genetic and biochemical approaches have been used to assign domains, functions, and sites within ~53. Domains of ~53 defined in terms of separable activities that contribute to its overall function include a transcrip tional activation region at the N-terminus (residues l-43) (Unger et al., 1992, and references therein); a sequencespecific DNA-binding domain (residues 100-300) (Bargonetti et al., 1993; Halazonetis and Kandil, 1993; Pavletich et al., 1993; Wang et al., 1993); an oligomerization domain that dictates that the protein form stable tetramers (residues 320-360) (Sturzbecher et al., 1992; Pavletich et al., 1993; Wang et al., 1993); and a nonspecific nucleic acidbinding (and reannealing) region (residues 330-393) (Wang et al., 1993, and references therein). Experiments with proteases have also been informative (Figure 1). ~53 has two protease-resistant domains: one within the central portion, extending roughly between
HPVFS-tqeteddepndpaw mlnimlmlanMromdugreglaeblon te-
DNA binding
, Core domain
C-tiIlIdWl domain
Minireview
Figure 1. Relationship to ~63 Landmarks
of Structural
Elements
The positions of the domains and protein interaction regions of ~53 are as indicated within and above the p53 sequence. Thevertical lines represent positions of tumor-derived p53 mutations. Heights of vertical lines reflect the relative frequency of mutation at each residue, with the six mutational hot spots indicated, taken from the p53 mutation data base (Lin et al., 1994; Hollstein et al., 1994). Above also are indicated the positions of defined phosphorylation sites, the candidate protein kinases, and nuclear localization signals (NLS). Below the sequence are protease (subtilisin)-cutting sites (Pavletich et al., 1993) indicated by arrows that define the core and C-terminal domains. The locations within the conserved regions of components of the two structural elements that are at the DNA-protein interface, the LSH and loops 2 and 3 as determined by X-ray crystallography(Choet al., 19, areindicated. Much of the scaffolding f3 sandwich is from the sequences between the conselved regions. The turn-6 strand-turn-a helix motif (TSTH) of a monomer of the p53 tetramer (Clore et al.. 1994) is shown below as well. Abbreviations: CDK, cyclindependent kinase; CKI. casein kinase I; CKII, casein kinase II; dsDNA, double stranded DNA; HPV, human papillomavirus; PK. protein kinase; TBP, TATA-binding protein.
amino acids 100 and 290, and a second one between amino acids 330 and 360, coinciding with the DNA-binding and tetramerization regions, respectively (Bargonetti et al., 1993; Pavlertich et al., 1993). The N-terminus and parts of the C-terminus are apparently more exposed to solvent and more sensitive to protease cutting (Pavletich et al., 1993). The absence of signals and phosphorylation sites within the central portion of the protein, which has a compactly folded and therefore relatively inaccessible structure, is consistent with the observations that most anti-p53 antibodies react with epftopes within the N- and C-termini rather than within the center (Legros et al., 1994, and references therein). The structures of the ~53 DNA-binding domain (Cho et al., 1994) and the oligomeritation region (Clore et al., 1994) have now been solved. Each structure has revealed itself to be highly novel and each has provided invaluable insight into the master switch that is ~53. The Structure of the Central Portion of ~53 Bound to DNA Pavletich and colleagues (Cho et al., 1994), using X-ray diffraction, determined the structure of the ~53 core-DNA cocrystal at a resolution of 2.2 A, refined to a crystallographic R factor of 20.5% (Figure 2). The cocrystal, including an oligomer that has one ~53 half-site 5’-GGGCAAGTCT-3’ and a weaker site one turn of the DNA away, contained two molecules of the core domain aligned as a head-to-tail dimer that corresponds to one half of the putative ~53 tetramer complex. A third core molecule was also detected that was not bound to DNA but was involved in crystal-packing contacts. Since the structures of both DNA-bound and nonbound cores are nearly identical, this suggests that the core does not undergo a structural alteration through binding to DNA. That the two DNA-bound
Figure 2. Ribbon Drawing Domain-DNA Complex
Depicting
the Structure
of the p53 Core
The DNA (blue) and the core domain (turquoise) are shown with the zinc atom (red), with the position of the six hot spot residues (yellow) indicated as well. Note that R243 and R273 directly contact DNA while the others are necessary for the integrity of the structural elements at the DNA-protein interface. Figure is taken from Cho et al. (1994) reprinted with permission from Science.
core molecules were aligned on the same face of the DNA has potential implications for regulation either by other regions of p53 or by other proteins interacting with the nonoccupied face. The ~53 DNA-binding domain, which is rather large when compared with others, revealed two major surprises. The first surprise is its novelty-the way in which the core recognizes its DNA sequence is essentially original. The domain consists of a6 sandwich, comprising two antiparallel 6 sheets, that serves as a scaffold for the structural elements at the DNA-protein interface. There are two components of these structural elements: a loop-sheethelix (LSH) motif that binds in the major groove and is involved in contacts with the bases, and two large loops, L2 and L3, that interact such that a critical residue, R246, which is in L3, makes contact with the minor groove of the DNA in the A/T-rich region of the binding site. As a result of its interaction with ~53, the minor groove of the DNA site is compressed, which may result in tight packing of R246 within this space. Virtually anything that affects L2 and L3 affects this critical R246 interaction. L2 and L3 interactions are stabilized by the zinc atom held in place by four metal-binding ligands, Cl76 and H179, which are in the L2 loop, and C236 and C242, within the L3 loop. The second surprise is that it is the four conserved regions, wherein the most frequently detected mutations lie, that comprise the L2 and L3 loops and the LSH motif that have critical roles in providing the structure, surfaces, and residues that actually contact the DNA (see Figure 1). Indeed, it is now possible to predict how each of the hot spot mutations alters or destabilizes the central domain such that it is no longer able to interact properly with cognate DNA sites. ~53 appears to be unique in that its DNA-binding region does not closely resemble other previously identified DNAbinding domain families, such as described by Pabo and Sauer (1992). Nevertheless, there are common features of all of these DNA-binding families that are shared by ~53. Side chain interactions occur (frequently involving hydrogen bonds) with both the bases and sugar-phosphate backbone of DNA. ~53 core amino acids make numerous hydrogen bonds with both the DNA backbone as well as two Gs and a C residue in the major groove. Likewise, as observed with other families, a ~53 a helix ( residues 276-266) fits within the major groove of the DNA. Additionally, although not in possession of a classical zinc finger, the tetrahedrally coordinated zinc atom is essential for the structure of the ~53 core. How do the tumor-derived mutations fit into the picture? This question is elegantly addressed by Cho et al. (1994) who carefully analyzed the structure in terms of the role of each of the hot spot amino acids. Based on their data, there are two classes of naturally occurring mutations in ~53: mutations in the first class directly interfere with protein-DNA phosphate or base contacts, while mutants of the second class have disrupted the structural integrity of the domain. These two classes can be distinguished by other criteria. The second but not the first class of mutants is more reactive with the antibody PAb 240 (Stephan and Lane, 1990), indicating exposure of a normally unavailable
Minireview 545
epitope within the hydrophobic core of the 8 sandwich; likewise, such mutants are much more protease sensitive (Bargonetti et al., 1993). Several of these mutations are well known; for example, Li-Fraumeni familial cancer patients frequently have a germline mutation at codon 248 (in fact, this codon is the most frequently mutated over all); likewise, there is a high occurrence of mutation at codon 249 in liver tumors in patients from areas where carcinogenic aflatoxins are dietary contaminants (Hollstein et al., 1994). While mutations at codon 248 and 273 affect direct interactions with DNA, mutations of codons 249 and 175 affect the overall structure of the core. R175 is involved in stabilizing interactions between loops 2 and 3 that are bridged by the zinc atom. Mutations at residue 175 are considered to be among the most disruptive to ~53 DNA-binding function. Thus, all naturally occurring mutations in p53 directly or indirectly affect the interaction of ~53 with DNA, demonstrating that sequence-specific DNA binding is central to the normal functioning of ~53 as a tumor suppressor. Nuclear Magnetic Resonance Structure of the p53 Oligomerization Domain However useful and important the insight derived from the structure of the ~53 core domain, it must be remembered that full-length ~53 exists as an oligomeric protein with sequences flanking the central DNA-binding region that are necessary for ~53 function. Both the full-length protein and the C-terminal portion of ~53 exist as tetramers in solution (Sturzbecher et al., 1992; Friedman et al., 1993; Wangetal., 1993). Human and murinep53canformstable heterotetramers, indicating that the oligomerization region is functionally conserved (Milner and Medcalf, 1991; Bargonetti et al., 1991). Clore et al. (1994) using multidimensional heteronuclear magnetic resonance spectroscopy, have solved the structure of a 42 amino acid peptide (residues 319-389) that contains the minimum tetramerization domain of human ~53. To determine the structure of this region, both 15N13C-labeled protein and protein consisting of equal amounts of unlabeled and labeled peptides were examined; in this way it was possible to determine the position of residues within each individual subunit as well as the relative position of the different subunits with respect to each other within the tetramer. Their data reveal that the structure of the highly symmetrical tetramer is quite novel. The monomeric unit contains a turn (residues 324-328), a 8 strand (residues 328-334) a second turn (residues 335-338), and an a helix (337-355). Each subunit interacts with another subunit such that the helices and 8 strands are antiparallel. Two such dimers interact, forming a four-helix bundle. The resulting tetramer is thus composed of a dimer of dimers. The interactions between the helices are stabilized by several defined salt bridges. What is the role of the tetramerization domain in p53? It is most easily speculated that it facilitates interactions with DNA. Indeed, although it is now well documented that ~53 levels do rise in response to DNA-damaging agents, the extent to which they do so is generally less than the comparable extent to which transcription from reporters bearing p53 response elements is stimulated. Thus, although it is not known how p53 finds its natural targets in
cells or how much ~53 is necessary for this, cooperative binding by ~53 monomers within a tetramer would greatly expedite interactions with ~53 response elements. The fact that the C-terminus of ~53 contains an autonomous nonspecific nucleic acid-binding region may be relevant in this respect as well. Why Are ~53 Mutations Clustered within its Center? The center of ~53 is indisputably essential for its tumor suppressor function. Nevertheless, without both activation and oligomerization regions, ~53 is dysfunctional in cells (Reed et al., 1993; Pietenpol et al., 1994). Yet there are few tumorderived mutations within these regions. Lin et al. (1994) generated an extensive series of single and multiple mutations within the acidic, proline-rich N-terminal activation domain. When they examined the resulting mutants’ abilities to activate transcription from templates bearing DNA-binding domains, they found that no single mutation within this region significantly affected transcrip tional activation by ~53. Furthermore, many multiple mutants, even one in which every one of the acidic residues was changed, were still capable of some transcriptional activation. This in itself is not surprising since the activation domains of many transcriptional activators are refractory to mutation. However, one (and only one) double mutant was identified, which changed two hydrophobic residues (L22 and W23) that displayed drastically reduced ~53 transcription function. That a ~53 gene will sustain two specific missense mutations is unlikely, which could explain why no such mutations have yet turned up in screens of tumor-derived ~53 genes. Only a very small number of missense mutations have been detected within the oligomerization domain. Nevertheless, as pointed out by Clore et al. (1994), each of these is likely to interfere with tetramerization, a prediction that remains to be tested. Future Directions The structures of the core domain-DNA cocrystal and the tetramerization region are likely to give scientists food for thought and motivation for experiments for some time to come. Additional structures may be solved that can add to our knowledge of ~53. For example, will it be possible to obtain structures of mutant p53 cores, and will they confirm the predictions of Cho et al. (1994)? What is the structure of the DNA-bound core linked to the tetramerization region? Is the DNA bent or wrapped by this version of p53? Hupp et al. (1993) have shown that the C-terminal 30 amino acids of ~53, a highly basic region, is capable of exerting a negative effect on ~53 DNA binding. Will the mode by which the core is repressed by the C-terminal region be deciphered through X-ray crystallography? Another compelling reason to study these structures is that they may be useful to identify wa$s to interfere with the role of ~53 in tumorigenesis. Wild-type ~53 induces apoptosis in some types of cells, including ones that have been subjected to radiation-induced DNA damage (Yonish-Rouach et al., 1991; Lowe et al., 1993; Clarke et al., 1993; Lee and Bernstein, 1993). By contrast, cells harboring mutant ~53 or lacking ~53 are frequently radiation resistant. Therefore, identifying nontoxic molecules that
would restore wild-type activity to mutant ~53 proteins in tumors (and thus induce the apoptotic pathway) holds the tantalizing promise of vastly extending the effectiveness of cancer therapy. The fact that the majority of codons in the sizable ~53 core domain have been mutated such that transcriptional activity is lost suggests that the overall structure of the core domain is important for its DNA-binding function and thus that this region may be very difficult to target for cancer therapy. However, all is not lost. Many hot spot mutant ~53 proteins have been shown to be inherently capable of binding to DNA. For example, forms of the protein that havelosttheextremeC-terminus(Huppet al., 1992,1993; Halazonetis and Kandil, 1993) display DNA binding. Other mutant p53 proteins bind DNA either at lower temperatures (e.g., Bargonetti et al., 1993) or to idealized p53binding sites (Zhang et al., 1993). Intensive screening programs, rational drug design approaches, or a combination of the two may yield the magic compounds that convert mutant to wild-type ~53 in cells. Refer8nce8 Bargonetti, J., Reynisdottir, Genes Dev. 6. 1886-1898. Bargonetti, C. (1993).
I., Friedman,
P. N., and Prives,
J., Manfredi, J. J., Chen, X., Marshak, Genes Dev. 12, 2565-2574.
Cho, Y.. Gorina. 265, 346-355.
S., Jeffrey,
P. C., and Pavletich,
Clarke, A. R., Purdie, C. A., Harrison, Hooper, M. L., and Wyllie, A. (1993).
C. (1991).
D. R., and Prives, N. P. (1994). Science
D. J.. Morris, R. G., Bird, C. C., Nature 362, 849-852.
Clore, G. M., Omichinski. J. G.. Sakaguchi, K., Zambrano, N., Sakamoto, H., Appella, E., and Gronenborn, A. M. (1994). Science 265, 386-391. Friedman, P. N., Chen, X., Bargonetti, Natl. Acad. Sci. USA 90. 3319-3323. Halazonetis, Holistein,
J., and Prives,
T. D., and Kandil, A. N. (1993). M., et al., (1994).
Hupp. T. Ft., Meek, 71( 875-006.
Nucl.
Acids
D. W., Midgley,
C. (1993).
Proc.
EMBO J. 72.5057-5964.
Res.,
in press.
C. A., and Lane, D. P. (1992).
Cell
Hupp, T. R., Meek, D. W., Midgley, Nucl. Acids Res. 21, 31679174.
C. A., and Lane,
D. P. (1993).
Lee, J. M., and Bernstein, 5742-5746.
Proc.
Sci. USA 90,
Legros,
Y.. Lafon,
Levine,
A. J. (1993).
Lin. J., Chen, 6, 1235-1246.
A. (1993).
C., and Soussi, Annu.
J., Elenbaas,
Natl. Acad.
T. (1994).
Oncogene 62, 623-651.
B., and Levine
A. J. (19Q4).
Lowe, S. W., Schmitt, E. M., Smith, T. (1993). Nature 362. 847-849.
S. E., Osbourne,
Milner,
Cell 65, 765-774.
J., and Medcalf,
Pabo, C. O., and Sauer, 1095.
E. (1991). R. T. (1992).
Pavtetich, N. P., Chambers, Dev. 72.2556-2564.
9, in press.
Rev. Biochem.
Annu.
Dev.
8. A., and Jacks,
Rev. Biochem.
K. A., and Pabo,
Genes
67,1053-
C. 0. (1993).
Genes
Pietenpol, J. A., Tokino, T., Thiagalingam, S., El-Deity, W. S., Kinzler, K. W., and Vogelstein, B. (1994). Proc. Natl. Aced. Sci. USA 97,19982002. Prives,
C., and Manfredi,
J. J. (1993).
Genes
Dev.
7, 529534.
Reed, M., Wang, Y., Mayr, G., Anderson, M. E., Schwedes, Tegtmeyer, P. (1993). Gene Expr. 3, 95-107. Stephan, Sturzbecher,
C. W., and Lane, D. P. (lQ99). H. W., Brain,
R., Addison,
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K., Remm,
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Grimatdi, M., Keenan, 1513-1523.
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J. R. (1992).
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August
26, 1994, Copyright
0 1994 by Cell Press
How Loops, p Sheets, and a Helices Help Us to Understand ~53 Carol Prives Department of Biological Sciences Columbia University New York, New York 10027
~53 plays a critical role in transducing a signal from damaged DNA to genes that control the cell cycle and apoptosis. Intrinsic to its function in this pathway is the ability of ~53, a tetrameric phosphoprotein, to bind sequence specifically to DNA. This property allows ~53 to serve as a transcriptional activator. Several ~53 target genes have been identified that contain a ~53 response element that conforms to the consensus sequence (5-R R R C AIT TIA G Y Y Y-33*. Tumor-derived ~53 mutants almost invariably display abnormal DNA binding and transcriptional activation properties. These and other relevant facts have been discussed in recent reviews (Levine, 1993; Prives and Manfredi, 1993). p53 Mutations in Cancer Experimental manipulation of the ~53 gene and protein has been useful for unveiling its functions and domains. However, there has been an equally powerful and revealing set of information about ~53 that has not been derived by experimentation-that resulting from sequencing the tumor-derived ~53 genes of nearly 2000 cancer patients (Figure 1; Lin et al., 1994; Hollstein et al., 1994). When aligned with the sequence of ~53 and compared with what is now known about its functions and properties, these
pSSbinding trnnseriplionalXlivPlion Ad Elb
SV4S T ml&en bindiq
TBP bind&
I
r
sequence-specific
mutants provide an extraordinary view of the ~53 protein. First, thevast majority of the mutationsare clustered within the central portion of the protein, where 4 of the 5 regions that are highly conserved among all ~53 genes sequenced to date are located. Second, although mutation of the great majority (but not all) of the amino acids within the center of p53 has occurred in tumors, there are among these a number of “hot spots” that occur with unusually high frequency. This spectrum of ~53 mutations tells us that central to the role of ~53 in preventing cancer is the very center of p53 itself. The Domains of p53 Reverse genetic and biochemical approaches have been used to assign domains, functions, and sites within ~53. Domains of ~53 defined in terms of separable activities that contribute to its overall function include a transcrip tional activation region at the N-terminus (residues l-43) (Unger et al., 1992, and references therein); a sequencespecific DNA-binding domain (residues 100-300) (Bargonetti et al., 1993; Halazonetis and Kandil, 1993; Pavletich et al., 1993; Wang et al., 1993); an oligomerization domain that dictates that the protein form stable tetramers (residues 320-360) (Sturzbecher et al., 1992; Pavletich et al., 1993; Wang et al., 1993); and a nonspecific nucleic acidbinding (and reannealing) region (residues 330-393) (Wang et al., 1993, and references therein). Experiments with proteases have also been informative (Figure 1). ~53 has two protease-resistant domains: one within the central portion, extending roughly between
HPVFS-tqeteddepndpaw mlnimlmlanMromdugreglaeblon te-
DNA binding
, Core domain
C-tiIlIdWl domain
Minireview
Figure 1. Relationship to ~63 Landmarks
of Structural
Elements
The positions of the domains and protein interaction regions of ~53 are as indicated within and above the p53 sequence. Thevertical lines represent positions of tumor-derived p53 mutations. Heights of vertical lines reflect the relative frequency of mutation at each residue, with the six mutational hot spots indicated, taken from the p53 mutation data base (Lin et al., 1994; Hollstein et al., 1994). Above also are indicated the positions of defined phosphorylation sites, the candidate protein kinases, and nuclear localization signals (NLS). Below the sequence are protease (subtilisin)-cutting sites (Pavletich et al., 1993) indicated by arrows that define the core and C-terminal domains. The locations within the conserved regions of components of the two structural elements that are at the DNA-protein interface, the LSH and loops 2 and 3 as determined by X-ray crystallography(Choet al., 19, areindicated. Much of the scaffolding f3 sandwich is from the sequences between the conselved regions. The turn-6 strand-turn-a helix motif (TSTH) of a monomer of the p53 tetramer (Clore et al.. 1994) is shown below as well. Abbreviations: CDK, cyclindependent kinase; CKI. casein kinase I; CKII, casein kinase II; dsDNA, double stranded DNA; HPV, human papillomavirus; PK. protein kinase; TBP, TATA-binding protein.
amino acids 100 and 290, and a second one between amino acids 330 and 360, coinciding with the DNA-binding and tetramerization regions, respectively (Bargonetti et al., 1993; Pavlertich et al., 1993). The N-terminus and parts of the C-terminus are apparently more exposed to solvent and more sensitive to protease cutting (Pavletich et al., 1993). The absence of signals and phosphorylation sites within the central portion of the protein, which has a compactly folded and therefore relatively inaccessible structure, is consistent with the observations that most anti-p53 antibodies react with epftopes within the N- and C-termini rather than within the center (Legros et al., 1994, and references therein). The structures of the ~53 DNA-binding domain (Cho et al., 1994) and the oligomeritation region (Clore et al., 1994) have now been solved. Each structure has revealed itself to be highly novel and each has provided invaluable insight into the master switch that is ~53. The Structure of the Central Portion of ~53 Bound to DNA Pavletich and colleagues (Cho et al., 1994), using X-ray diffraction, determined the structure of the ~53 core-DNA cocrystal at a resolution of 2.2 A, refined to a crystallographic R factor of 20.5% (Figure 2). The cocrystal, including an oligomer that has one ~53 half-site 5’-GGGCAAGTCT-3’ and a weaker site one turn of the DNA away, contained two molecules of the core domain aligned as a head-to-tail dimer that corresponds to one half of the putative ~53 tetramer complex. A third core molecule was also detected that was not bound to DNA but was involved in crystal-packing contacts. Since the structures of both DNA-bound and nonbound cores are nearly identical, this suggests that the core does not undergo a structural alteration through binding to DNA. That the two DNA-bound
Figure 2. Ribbon Drawing Domain-DNA Complex
Depicting
the Structure
of the p53 Core
The DNA (blue) and the core domain (turquoise) are shown with the zinc atom (red), with the position of the six hot spot residues (yellow) indicated as well. Note that R243 and R273 directly contact DNA while the others are necessary for the integrity of the structural elements at the DNA-protein interface. Figure is taken from Cho et al. (1994) reprinted with permission from Science.
core molecules were aligned on the same face of the DNA has potential implications for regulation either by other regions of p53 or by other proteins interacting with the nonoccupied face. The ~53 DNA-binding domain, which is rather large when compared with others, revealed two major surprises. The first surprise is its novelty-the way in which the core recognizes its DNA sequence is essentially original. The domain consists of a6 sandwich, comprising two antiparallel 6 sheets, that serves as a scaffold for the structural elements at the DNA-protein interface. There are two components of these structural elements: a loop-sheethelix (LSH) motif that binds in the major groove and is involved in contacts with the bases, and two large loops, L2 and L3, that interact such that a critical residue, R246, which is in L3, makes contact with the minor groove of the DNA in the A/T-rich region of the binding site. As a result of its interaction with ~53, the minor groove of the DNA site is compressed, which may result in tight packing of R246 within this space. Virtually anything that affects L2 and L3 affects this critical R246 interaction. L2 and L3 interactions are stabilized by the zinc atom held in place by four metal-binding ligands, Cl76 and H179, which are in the L2 loop, and C236 and C242, within the L3 loop. The second surprise is that it is the four conserved regions, wherein the most frequently detected mutations lie, that comprise the L2 and L3 loops and the LSH motif that have critical roles in providing the structure, surfaces, and residues that actually contact the DNA (see Figure 1). Indeed, it is now possible to predict how each of the hot spot mutations alters or destabilizes the central domain such that it is no longer able to interact properly with cognate DNA sites. ~53 appears to be unique in that its DNA-binding region does not closely resemble other previously identified DNAbinding domain families, such as described by Pabo and Sauer (1992). Nevertheless, there are common features of all of these DNA-binding families that are shared by ~53. Side chain interactions occur (frequently involving hydrogen bonds) with both the bases and sugar-phosphate backbone of DNA. ~53 core amino acids make numerous hydrogen bonds with both the DNA backbone as well as two Gs and a C residue in the major groove. Likewise, as observed with other families, a ~53 a helix ( residues 276-266) fits within the major groove of the DNA. Additionally, although not in possession of a classical zinc finger, the tetrahedrally coordinated zinc atom is essential for the structure of the ~53 core. How do the tumor-derived mutations fit into the picture? This question is elegantly addressed by Cho et al. (1994) who carefully analyzed the structure in terms of the role of each of the hot spot amino acids. Based on their data, there are two classes of naturally occurring mutations in ~53: mutations in the first class directly interfere with protein-DNA phosphate or base contacts, while mutants of the second class have disrupted the structural integrity of the domain. These two classes can be distinguished by other criteria. The second but not the first class of mutants is more reactive with the antibody PAb 240 (Stephan and Lane, 1990), indicating exposure of a normally unavailable
Minireview 545
epitope within the hydrophobic core of the 8 sandwich; likewise, such mutants are much more protease sensitive (Bargonetti et al., 1993). Several of these mutations are well known; for example, Li-Fraumeni familial cancer patients frequently have a germline mutation at codon 248 (in fact, this codon is the most frequently mutated over all); likewise, there is a high occurrence of mutation at codon 249 in liver tumors in patients from areas where carcinogenic aflatoxins are dietary contaminants (Hollstein et al., 1994). While mutations at codon 248 and 273 affect direct interactions with DNA, mutations of codons 249 and 175 affect the overall structure of the core. R175 is involved in stabilizing interactions between loops 2 and 3 that are bridged by the zinc atom. Mutations at residue 175 are considered to be among the most disruptive to ~53 DNA-binding function. Thus, all naturally occurring mutations in p53 directly or indirectly affect the interaction of ~53 with DNA, demonstrating that sequence-specific DNA binding is central to the normal functioning of ~53 as a tumor suppressor. Nuclear Magnetic Resonance Structure of the p53 Oligomerization Domain However useful and important the insight derived from the structure of the ~53 core domain, it must be remembered that full-length ~53 exists as an oligomeric protein with sequences flanking the central DNA-binding region that are necessary for ~53 function. Both the full-length protein and the C-terminal portion of ~53 exist as tetramers in solution (Sturzbecher et al., 1992; Friedman et al., 1993; Wangetal., 1993). Human and murinep53canformstable heterotetramers, indicating that the oligomerization region is functionally conserved (Milner and Medcalf, 1991; Bargonetti et al., 1991). Clore et al. (1994) using multidimensional heteronuclear magnetic resonance spectroscopy, have solved the structure of a 42 amino acid peptide (residues 319-389) that contains the minimum tetramerization domain of human ~53. To determine the structure of this region, both 15N13C-labeled protein and protein consisting of equal amounts of unlabeled and labeled peptides were examined; in this way it was possible to determine the position of residues within each individual subunit as well as the relative position of the different subunits with respect to each other within the tetramer. Their data reveal that the structure of the highly symmetrical tetramer is quite novel. The monomeric unit contains a turn (residues 324-328), a 8 strand (residues 328-334) a second turn (residues 335-338), and an a helix (337-355). Each subunit interacts with another subunit such that the helices and 8 strands are antiparallel. Two such dimers interact, forming a four-helix bundle. The resulting tetramer is thus composed of a dimer of dimers. The interactions between the helices are stabilized by several defined salt bridges. What is the role of the tetramerization domain in p53? It is most easily speculated that it facilitates interactions with DNA. Indeed, although it is now well documented that ~53 levels do rise in response to DNA-damaging agents, the extent to which they do so is generally less than the comparable extent to which transcription from reporters bearing p53 response elements is stimulated. Thus, although it is not known how p53 finds its natural targets in
cells or how much ~53 is necessary for this, cooperative binding by ~53 monomers within a tetramer would greatly expedite interactions with ~53 response elements. The fact that the C-terminus of ~53 contains an autonomous nonspecific nucleic acid-binding region may be relevant in this respect as well. Why Are ~53 Mutations Clustered within its Center? The center of ~53 is indisputably essential for its tumor suppressor function. Nevertheless, without both activation and oligomerization regions, ~53 is dysfunctional in cells (Reed et al., 1993; Pietenpol et al., 1994). Yet there are few tumorderived mutations within these regions. Lin et al. (1994) generated an extensive series of single and multiple mutations within the acidic, proline-rich N-terminal activation domain. When they examined the resulting mutants’ abilities to activate transcription from templates bearing DNA-binding domains, they found that no single mutation within this region significantly affected transcrip tional activation by ~53. Furthermore, many multiple mutants, even one in which every one of the acidic residues was changed, were still capable of some transcriptional activation. This in itself is not surprising since the activation domains of many transcriptional activators are refractory to mutation. However, one (and only one) double mutant was identified, which changed two hydrophobic residues (L22 and W23) that displayed drastically reduced ~53 transcription function. That a ~53 gene will sustain two specific missense mutations is unlikely, which could explain why no such mutations have yet turned up in screens of tumor-derived ~53 genes. Only a very small number of missense mutations have been detected within the oligomerization domain. Nevertheless, as pointed out by Clore et al. (1994), each of these is likely to interfere with tetramerization, a prediction that remains to be tested. Future Directions The structures of the core domain-DNA cocrystal and the tetramerization region are likely to give scientists food for thought and motivation for experiments for some time to come. Additional structures may be solved that can add to our knowledge of ~53. For example, will it be possible to obtain structures of mutant p53 cores, and will they confirm the predictions of Cho et al. (1994)? What is the structure of the DNA-bound core linked to the tetramerization region? Is the DNA bent or wrapped by this version of p53? Hupp et al. (1993) have shown that the C-terminal 30 amino acids of ~53, a highly basic region, is capable of exerting a negative effect on ~53 DNA binding. Will the mode by which the core is repressed by the C-terminal region be deciphered through X-ray crystallography? Another compelling reason to study these structures is that they may be useful to identify wa$s to interfere with the role of ~53 in tumorigenesis. Wild-type ~53 induces apoptosis in some types of cells, including ones that have been subjected to radiation-induced DNA damage (Yonish-Rouach et al., 1991; Lowe et al., 1993; Clarke et al., 1993; Lee and Bernstein, 1993). By contrast, cells harboring mutant ~53 or lacking ~53 are frequently radiation resistant. Therefore, identifying nontoxic molecules that
would restore wild-type activity to mutant ~53 proteins in tumors (and thus induce the apoptotic pathway) holds the tantalizing promise of vastly extending the effectiveness of cancer therapy. The fact that the majority of codons in the sizable ~53 core domain have been mutated such that transcriptional activity is lost suggests that the overall structure of the core domain is important for its DNA-binding function and thus that this region may be very difficult to target for cancer therapy. However, all is not lost. Many hot spot mutant ~53 proteins have been shown to be inherently capable of binding to DNA. For example, forms of the protein that havelosttheextremeC-terminus(Huppet al., 1992,1993; Halazonetis and Kandil, 1993) display DNA binding. Other mutant p53 proteins bind DNA either at lower temperatures (e.g., Bargonetti et al., 1993) or to idealized p53binding sites (Zhang et al., 1993). Intensive screening programs, rational drug design approaches, or a combination of the two may yield the magic compounds that convert mutant to wild-type ~53 in cells. Refer8nce8 Bargonetti, J., Reynisdottir, Genes Dev. 6. 1886-1898. Bargonetti, C. (1993).
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