Some 50% of human cancers are associated with mutations in the core domain of the tumor suppressor p53. Many mutations are thought just to destabilize the protein. To assess this and the possibility of rescue, we have set up a system to analyze the stability of the core domain and its mutants. The use of differential scanning calorimetry or spectroscopy to measure its melting temperature leads to irreversible denaturation and aggregation and so is useful as only a qualitative guide to stability. There are excellent two-state denaturation curves on the addition of urea that may be analyzed quantitatively. One Zn 2؉ ion remains tightly bound in the holo-form of p53 throughout the denaturation curve. The stability of wild type is 6.0 kcal (1 kcal ‫؍‬ 4.18 kJ)͞mol at 25°C and 9.8 kcal͞mol at 10°C. The oncogenic mutants R175H, C242S, R248Q, R249S, and R273H are destabilized by 3.0, 2.9, 1.9, 1.9, and 0.4 kcal͞mol, respectively. Under certain denaturing conditions, the wildtype domain forms an aggregate that is relatively highly f luorescent at 340 nm on excitation at 280 nm. The destabilized mutants give this f luorescence under milder denaturation conditions.The tumor suppressor protein p53 is a sequence-specific transcription factor that functions to maintain the integrity of the genome (1). On its induction in response to DNA damage, p53 promotes cell cycle arrest in G 1 phase (2) and apoptosis if DNA repair is not possible (3). Negative regulation occurs by the synthesis and subsequent binding of the oncoprotein Mdm2 to the transactivation domain of p53. This targets it for degradation and ensures that the cellular stability of p53 is low (4, 5). About 50% of human cancers and 95% of lung cancers are associated with mutations in p53. The majority of these map to its core domain, which is responsible for binding DNA (6). The crystal structure of the core domain bound to DNA has been determined (7). A number of the tumorigenic mutants affect residues that contact the DNA, but many are not directly involved in binding and appear to affect the thermodynamic stability of the protein (8, 9). p53 is a possible target for cancer therapy, including drugs that can stabilize it or using superstable p53 variants that would be suitable for gene therapy applications. There is a lack of quantitative information on the stability of p53 on which to base experiments measuring its change in stability on mutation. Data tend to be restricted so far to measurements of the temperature dependence of transactivation or PAb 1620 binding (8, 9), a monoclonal antibody specific for the native state of wild-type p53 (10). These suggest that p53 is relatively unstable. We find in this study that the core domain denatures irreversibly with temperature, and so the T m measured by differential scanning calorimetry or spectroscopy cannot be used quantitatively for analyzing structure-activity relationships of p53. We have turned instead to studying the stability of the isolated core domain by using urea-mediated denaturation, which is of p...

Most of the oncogenic mutations in the tumor suppressor p53 map to its DNA-binding (core) domain. It is thus a potential target in cancer therapy for rescue by drugs. To begin to understand how mutation inactivates p53 and hence to provide a structural basis for drug design, we have compared structures of wild-type and mutant p53 core domains in solution by NMR spectroscopy. Structural changes introduced by five hot-spot mutations (V143A, G245S, R248Q, R249S, and R273H) were monitored by chemical-shift changes. Only localized changes are observed for G245S, R248Q, R249S, and R273H, suggesting that the overall tertiary folds of these mutant proteins are similar to that of wild type. Structural changes in R273H are found mainly in the loop-sheet-helix motif and the loop L3 of the core domain. Mutations in L3 (G245S, R248Q, and R249S) introduce structural changes in the loop L2 and L3 as well as terminal residues of strands 4, 9, and 10. It is noteworthy that R248Q, which is often regarded as a contact mutant that affects only interactions with DNA, introduces structural changes as extensive as the other loop L3 mutations (G245S and R249S). These changes suggest that R248Q is also a structural mutant that perturbs the structure of loop L2-L3 regions of the p53 core domain. In contrast to other mutants, replacement of the core residue valine 143 to alanine causes chemical-shift changes in almost all residues in the ␤-sandwich and the DNA-binding surface. Long-range effects of V143A mutation may affect the specificity of DNA binding.

The core domain of p53 is extremely susceptible to mutations that lead to loss of function. We analysed the stability and DNA-binding activity of such mutants to understand the mechanism of second-site suppressor mutations. Double-mutant cycles show that N239Y and N268D act as 'global stability' suppressors by increasing the stability of the cancer mutants G245S and V143A-the free energy changes are additive. Conversely, the suppressor H168R is specific for the R249S mutation: despite destabilizing wild type, H168R has virtually no effect on the stability of R249S, but restores its binding affinity for the gadd45 promoter. NMR structural comparisons of R249S/H168R and R249S/T123A/H168R with wild type and R249S show that H168R reverts some of the structural changes induced by R249S. These results have implications for possible drug therapy to restore the function of tumorigenic mutants of p53: the function of mutants such as V143A and G245S is theoretically possible to restore by small molecules that simply bind to and hence stabilize the native structure, whereas R249S requires alteration of its mutant native structure.

This study analyzes the three-dimensional structure of the TATA-box properties. The crystal structure of this hyperthermostable protein was Cambridge University, compared to its mesophilic homologs and analyzed for differences in the Cambridge, CB2 1QR, UK native structure that may contribute to thermostability. Differences found were: (1) a disulfide bond not found in mesophilic counterparts; (2) an increased number of surface electrostatic interactions; (3) more compact protein packing. The presumed DNA binding surface of PwTBP, like its eukaryotic counterparts, is hydrophobic but the electrostatic profile surrounding the protein is relatively neutral compared to the asymmetric positive potential that surrounds eukaryotic TBPs. The total reliance on a hydrophobic interface with DNA may explain the enhanced affinity of PwTBP for its DNA promoter at higher temperatures and increased salt concentration.

Archaea possess a basal transcriptional apparatus that resembles that of eukaryotes. Here we report the 2.1-Å crystal structure of the archaeal transcription factor complex formed by the TATA-box-binding protein (TBP), the transcription factor IIB homolog, and a DNA target, all from the hyperthermophile Pyrococcus woesei. The overall fold of these two basal transcription factors is essentially the same as that of their eukaryotic counterparts. However, in comparison with the eukaryotic complexes, the archaeal TBP-DNA interface is more symmetrical, and in this structure the orientation of the preinitiation complex assembly on the promoter is inverted with respect to that seen in all crystal structures of comparable eukaryotic systems. This study of the structural details of an archaeal transcription factor complex presents the opportunity to examine the evolution of the basal eukaryotic transcriptional apparatus from a stereochemical viewpoint and to extend our understanding of the physical biochemistry of transcriptional initiation.The recent sequencing of the first archaeal genome has confirmed the designation of the archaea as a third kingdom of life, distinct from eubacteria and eukaryotes (1). Recent discoveries indicate that the basal components of transcription in archaea resemble those in the eukaryotic RNA polymerase II transcriptional system (2). Archaeal homologs to the transcription factors TATA-box binding protein (TBP) and transcription factor IIB (TFIIB) have been reported (3-6). As in eukaryotic polymerase II transcription, archaeal transcription is initiated by an AϩT-rich TATA-like segment known as the "boxA" sequence, containing a consensus T C TTA T A ANN (hereafter referred to as the boxA͞TATA element) (7,8). Interestingly, one of the common archaeal promoter sequences contains the boxA͞TATA element, TTTATATA, which is an inverted canonical eukaryotic TATA box (8). Like eukaryotic TBP, archaeal TBP recognizes the boxA͞TATA element, and transcription factor B (TFB) from Pyrococcus woesei (pwTFB) binds to the TBP-DNA complex (3). Homologs to other eukaryotic basal transcription factors, or TBP-associated factors (TAFs), however, are not present in the archaeal genome. In fact, promoter-specific transcription can occur in a purified reconstituted archaeal system, with only TBP, TFB, and polymerase (9).We present here the 2.1-Å crystal structure of the ternary archaeal transcription complex, illustrating the interaction of TBP, TFB, and a promoter fragment. The components are all from P. woesei, a hyperthermophilic archaeon which exhibits an optimal growth temperature of 105°C (10). The amino acid sequence of pwTBP is 36-41% identical to the conserved C-terminal core of its eukaryotic counterparts (3), and, as expected from this degree of homology, we have recently found that the general features of its three-dimensional structure are the same as those of eukaryotic TBP (11). pwTFB is 28-32% identical to full-length eukaryotic TFIIBs, and 25% identical to the C-terminal domain of the yeas...

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Semiempirical molecular orbital calculations are combined with 13C NMR chemical shifts to localize the counterion in the retinal binding site of vertebrate rhodopsin. Charge densities along the polyene chain are calculated for an 11-cis-retinylidene protonated Schiff base (11-cis-RPSB) chromophore with 1) a chloride counterion at various distances from the Schiff base nitrogen, 2) one or two chloride counterions at different positions along the retinal chain from C10 to C15 and at the Schiff base nitrogen, and 3) a carboxylate counterion out of the retinal plane near C12. Increasing the distance of the negative counterion from the Schiff base results in an enhancement of alternating negative and positive partial charge on the even- and odd-numbered carbons, respectively, when compared to the 11-cis-RPSB chloride model compound. In contrast, the observed 13C NMR data of rhodopsin exhibit downfield chemical shifts from C8 to C13 relative to the 11-cis-RPSB.Cl corresponding to a net increase of partial positive or decrease of partial negative charge at these positions (Smith, S. O., I. Palings, M. E. Miley, J. Courtin, H. de Groot, J. Lugtenburg, R. A. Mathies, and R. G. Griffin. 1990. Biochemistry. 29:8158-8164). The anomalous changes in charge density reflected in the rhodopsin NMR chemical shifts can be qualitatively modeled by placing a single negative charge above C12. The calculated fit improves when a carboxylate counterion is used to model the retinal binding site. Inclusion of water in the model does not alter the fit to the NMR data, although it is consistent with observations based on other methods. These data constrain the location and the orientation of the Glu113 side chain, which is known to be the counterion in rhodopsin, and argue for a strong interaction centered at C12 of the retinylidene chain.

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