Temperature-dependent switching between "wild-type" and "mutant" forms of p53-Val135

The p53 gene is a suppressor of abnormal cell growth but is also subject to oncogenic activation by mutation. The mutant allele p53-Val135, has recently been discovered to be temperature-sensitive and functions as an oncogene at 37 degrees C and as a tumor suppressor at 32.5 degrees C. In order to investigate the molecular mechanism underlying the temperature sensitivity of p53-Val135 rabbit reticulocyte lysate was used to translate the p53 mRNAs in vitro at 37 degrees C and at 30 degrees C. The immunoreactivity and T antigen binding of wild-type protein p53-Ala135 were unaffected by temperature and were similar to wild-type p53 expressed in vivo. In contrast, the mutant p53-Val135 protein was markedly affected by temperature. At 37 degrees C p53-Val135 showed reduced T antigen binding and did not react with monoclonal antibodies PAb246 and PAb1620. At 30 degrees C, p53-Val135 behaved as the wild-type p53. Temperature also exerted a post-translational effect on p53-Val135 with complete conversion from wild-type to mutant phenotype within two minutes of temperature shift from 30 degrees C to 37 degrees C. There was incomplete conversion from mutant to wild-type phenotype when the temperature was shifted down from 37 degrees C to 30 degrees C. We propose that the temperature dependent forms of p53-Val135 represent conformational variants of the p53 protein with opposing functions in cell growth control.     

Tolerance of a protein to multiple polar-to-hydrophobic surface substitutions

Hydrophobic substitutions at solvent-exposed positions in two alpha-helical regions of the bacteriophage P22 Arc repressor were introduced by combinatorial mutagenesis. In helix A, hydrophobic residues were tolerated individually at each of the five positions examined, but multiple substitutions were poorly tolerated as shown by the finding that mutants with more than two additional hydrophobic residues were biologically inactive. Several inactive helix A variants were purified and found to have reduced thermal stability relative to wild-type Arc, with a rough correlation between the number of polar-to-hydrophobic substitutions and the magnitude of the stability defect. Quite different results were obtained in helix B, where variants with as many as five polar-to-hydrophobic substitutions were found to be biologically active and one variant with three hydrophobic substitutions had a t(m) 6 degrees C higher than wild-type. By contrast, a helix A mutant with three similar polar-to-hydrophobic substitutions was 23 degrees C less stable than wild-type. Also, one set of three polar-to-hydrophobic substitutions in helix B was tolerated when introduced into the wild-type background but not when introduced into an equally active mutant having a nearly identical structure. Context effects occur both when comparing different regions of the same protein and when comparing the same region in two different homologues.     

Tolerance of T4 lysozyme to proline substitutions within the long interdomain alpha-helix illustrates the adaptability of proteins to potentially destabilizing lesions.

To investigate the ability of a protein to accommodate potentially destabilizing amino acid substitutions, and also to investigate the steric requirements for catalysis, proline was substituted at different sites within the long alpha-helix that connects the amino-terminal and carboxyl-terminal domains of T4 lysozyme. Of the four substitutions attempted, three yielded folded, functional proteins. The catalytic activities of these three mutant proteins (Q69P, D72P, and A74P) were 60-90% that of wild-type. Their melting temperatures were 7-12 degrees C less than that of wild-type at pH 6.5. Mutant D72P formed crystals isomorphous with wild-type allowing the structure to be determined at high resolution. In the crystal structure of wild-type lysozyme the interdomain alpha-helix has an overall bend angle of 8.5 degrees. In the mutant structure the introduction of the proline causes this bend angle to increase to 14 degrees and also causes a corresponding rotation of 5.5 degrees of carboxyl-terminal domain relative to the amino-terminal one. Except for the immediate location of the proline substitution there is very little change in the geometry of the interdomain alpha-helix. The results support the view that protein structures are adaptable and can compensate for potentially destabilizing amino acid substitutions. The results also suggest that the precise shape of the active site cleft of T4 lysozyme is not critical for catalysis.     

Thermal stabilities of mutant Escherichia coli tryptophan synthase alpha subunits.

Random chemical mutagenesis, in vitro, of the 5' portion of the Escherichia coli trpA gene has yielded 66 mutant alpha subunits containing single amino acid substitutions at 49 different residue sites within the first 121 residues of the protein; this portion of the alpha subunit contains four of the eight alpha helices and three of the eight beta strands in the protein. Sixty-two of the subunits were examined for their heat stabilities by sensitivity to enzymatic inactivation (52 degrees C for 20 min) in crude extracts and by differential scanning calorimetry (DSC) with 29 purified proteins. The enzymatic activities of mutant alpha subunits that contained amino acid substitutions within the alpha and beta secondary structures were more heat labile than the wild-type alpha subunit. Alterations only in three regions, at or immediately C-terminal to the first three beta strands, were stability neutral or stability enhancing with respect to enzymatic inactivation. Enzymatic thermal inactivation appears to be correlated with the relative accessibility of the substituted residues; stability-neutral mutations are found at accessible residual sites, stability-enhancing mutations at buried sites. DSC analyses showed a similar pattern of stabilization/destabilization as indicated by inactivation studies. Tm differences from the wild-type alpha subunit varied +/- 7.6 degrees C. Eighteen mutant proteins containing alterations in helical and sheet structures had Tm's significantly lower (-1.6 to -7.5 degrees C) than the wild-type Tm (59.5 degrees C). In contrast, 6 mutant alpha subunits with alterations in the regions following beta strands 1 and 3 had increased Tm's (+1.4 to +7.6 degrees C). Because of incomplete thermal reversibilities for many of the mutant alpha subunits, most likely due to identifiable aggregated forms in the unfolded state, reliable differences in thermodynamic stability parameters are not possible. The availability of this group of mutant alpha subunits which clearly contain structural alterations should prove useful in defining the roles of certain residues or sequences in the unfolding/folding pathway for this protein when examined by urea/guaninidine denaturation kinetic analysis.     

Role of the Cro repressor carboxy-terminal domain and flexible dimer linkage in operator and nonspecific DNA binding

A series of mutations comprising single and multiple substitutions, deletions, and extensions within the carboxy-terminal domain of the bacteriophage lambda Cro repressor have been constructed. These mutations generally affect the affinity of repressor for specific and nonspecific DNA. Additionally, substitution of the carboxy-terminal alanine with several amino acids capable of hydrogen-bonding interactions leads to improved specific binding affinities. A mutation is also described whereby cysteine links the two Cro monomers by a disulfide bond. As a consequence, a significant improvement in nonspecific binding and a concomitant reduction in specific binding are observed with this mutant. These results provide evidence that the carboxy terminus of Cro repressor is an important DNA binding domain and that a flexible connection between the two repressor monomers is a critical factor in modulating the affinity of wild-type repressor for DNA.     

Domains in lambda Cro repressor. A calorimetric study.

Thermodynamic properties of a mutant lambda Cro repressor with Cys replacing Val55 were studied calorimetrically. Formation of the S-S cross-link between neighboring Cys55 residues in this dimeric molecule leads to stabilization of a structure formed by the C-terminal parts of the two polypeptide chains, which behave as a single cooperative domain upon protein denaturation by heating. This composite domain is very stable at neutral pH and disrupts at 110 degrees C. The S-S-cross-linked tryptic fragment (residues 22-66), which includes this C-terminal domain, has similar stability. The N-terminal parts of the polypeptide chains do not form any stable structure when isolated, but in S-S-cross-linked dimer, they form a single cooperative block which melts in an all-or-none way 9 degrees C higher than the un-cross-linked protein. The observed cooperation of the distant N-terminal parts in dimer raises questions regarding lambda Cro repressor structure in solution.     

Change in oligomerization specificity of the p53 tetramerization domain by hydrophobic amino acid substitutions.

The tumor suppressor function of the wild-type p53 protein is transdominantly inhibited by tumor-derived mutant p53 proteins. Such transdominant inhibition limits the prospects for gene therapy approaches that aim to introduce wild-type p53 into cancer cells. The molecular mechanism for transdominant inhibition involves sequestration of wild-type p53 subunits into inactive wild-type/mutant hetero-tetramers. Thus, p53 proteins, whose oligomerization specificity is altered so they cannot interact with tumor-derived mutant p53, would escape transdominant inhibition. Aided by the known three-dimensional structure of the p53 tetramerization domain and by trial and error we designed a novel domain with seven amino acid substitutions in the hydrophobic core. A full-length p53 protein bearing this novel domain formed homo-tetramers and had tumor suppressor function, but did not hetero-oligomerize with tumor-derived mutant p53 and resisted transdominant inhibition. Thus, hydrophobic core residues influence the oligomerization specificity of the p53 tetramerization domain.     

The design of a hyperstable mutant of the Abp1p SH3 domain by sequence alignment analysis

We have characterized the thermodynamic stability of the SH3 domain from the Saccharomyces cerevisiae Abp1p protein and found it to be relatively low compared to most other SH3 domains, with a Tm of 60 degrees C and a deltaGu of 3.08 kcal/mol. Analysis of a large alignment of SH3 domains led to the identification of atypical residues at eight positions in the wild-type Abp1p SH3 domain sequence that were subsequently replaced by the residue seen most frequently at that position in the alignment. Three of the eight mutants constructed in this way displayed increases in Tm ranging from 8 to 15 degrees C with concomitant increases in deltaGu of up to 1.4 kcal/mol. The effects of these substitutions on folding thermodynamics and kinetics were entirely additive, and a mutant containing all three was dramatically stabilized with a Tm greater than 90 degrees C and a deltaGu more than double that of the wild-type domain. The folding rate of this hyperstable mutant was 10-fold faster than wild-type, while its unfolding rate was fivefold slower. All of the stabilized mutants were still able to bind a target peptide with wild-type affinity. We have analyzed the stabilizing amino acid substitutions isolated in this study and several other similar sequence alignment based studies. In approximately 25% of cases, increased stability can be explained by enhanced propensity of the substituted residue for the local backbone conformation at the mutagenized site.     

Identification, cloning, and characterization of the Escherichia coli sohA gene, a suppressor of the htrA (degP) null phenotype.

Two extragenic suppressors which allow temperature-sensitive htrA mutant Escherichia coli bacteria to grow at 42 degrees C and simultaneously acquire a cold-sensitive phenotype at 30 degrees C were isolated. The cold-sensitive phenotype exhibited by one of the mutants was used to clone the corresponding wild-type copy of the suppressor gene. This was done through complementation with a mini-mu plasmid E. coli DNA library, by selection for colonies which were no longer cold sensitive, at 30 degrees C. The cloned suppressor gene was shown to complement the cold-sensitive phenotype of both suppressor mutations. It was mapped to 68 min on the E. coli chromosome through hybridization to the Kohara library of overlapping lambda transducing bacteriophages, which covers the entire E. coli chromosome. The complementing gene was further subcloned on an 830-base-pair (bp) DNA fragment. DNA sequencing revealed the presence of an open reading frame (ORF) of 333 bp which could encode a protein of 12,359 Mr. Subcloning of various DNA fragments from within this 830-bp DNA fragment suggests that this ORF is most likely responsible for suppression of the cold-sensitive phenotype of the htrA suppressor bacteria. By using a T7 polymerase system to overproduce plasmid-encoded proteins, a protein of approximately 12,000 Mr was produced by this cloned DNA fragment. This ORF defines a previously undiscovered gene in E. coli, called sohA (suppressor of htrA).     

Molecular properties of global suppressors of temperature-sensitive folding mutations in P22 tailspike endorhamnosidase.

Two global suppressors (Val-331 greater than Ala and Ala-334 greater than Val) have been identified for temperature-sensitive folding (tsf) mutations in gene 9 of bacteriophage P22 (Mitraki, A., Fane, B., Haase-Pettingell, C., Sturtevant, J., and King, J. (1991) Science 253, 54-58). We have introduced 19 different single amino acid substitutions at the two global suppressor sites independently and examined the effects on the tailspike formation in Escherichia coli. Folding and maturation patterns of the various substitutions at the two global suppressor sites in the wild-type background suggest that Val-331 is located on the protein surface and Ala-334 is in the hydrophobic region. In combination with a tsf mutation, tsfH304 (Gly-244 greater than Arg), only Gly at 331 and Ile at 334, the substitutions that have similar side chain properties to the original suppressor sequences, were active as tsf suppressors. The newly identified suppressors of tsfH304 could also alleviate the tsf defect of three other mutations. The mutant carrying both Val-331 greater than Ala and Ala-334 greater than Val substitutions was also a global suppressor and was more active in suppressing the tsf defect than mutants carrying only one substitution. The suppressors may act by increasing the stability of an intermediate in the productive pathway of folding and maturation of the mutant polypeptides.     

Effect of single amino acid substitutions on the protease susceptibility of tryptophan synthase alpha subunit

In order to explore the correlation between protease susceptibility and conformational stability of a protein, the proteolytic degradation by trypsin, subtilisin and pronase P of the wild-type alpha subunit of tryptophan synthase from Escherichia coli and of its two mutant proteins was studied by measuring circular dichroism at 222 nm at various pH values at 37 degrees C. The mutant proteins are substituted by Gln or Met in place of Glu at position 49. The single amino acid substitutions at position 49 significantly affected susceptibility of this protein to the three proteases. Dependence of protease susceptibility of the wild-type and the two mutant proteins on pH was characteristic of each protein and similar for the three proteases. Comparison of the present results with the conformational stabilities of the three proteins previously measured shows that the order of resistance to the proteases among the three proteins coincides with the order of the values of unfolding Gibbs energy change, suggesting that protein degradation depends upon the conformational stability of a protein.     

Interaction between FliE and FlgB, a proximal rod component of the flagellar basal body of Salmonella

FliE is a flagellar basal body protein of Salmonella whose detailed location and function have not been established. A mutant allele of fliE, which caused extremely poor flagellation and swarming, generated extragenic suppressors, all of which mapped to flgB, one of four genes encoding the basal body rod; the fliE flgB pseudorevertants were better flagellated and swarmed better than the fliE parent, especially when the temperature was reduced from 37 to 30 degrees C. Motility of the pseudorevertants in liquid culture was markedly better than motility on swarm plates; we interpret this to mean that reduced flagellation is less deleterious at low viscous loads. Overproduction of the mutant FliE protein improved the motility of the parental fliE mutant and its pseudorevertants, though not to wild-type levels. Overproduction of suppressor FlgB (but not wild-type FlgB) in the fliE mutant also resulted in improved motility. The second-site FlgB mutation by itself had no phenotype; cells swarmed as well as wild-type cells. When overproduced, wild-type FliE was dominant over FliE-V99G, but the reverse was not true; that is, overproduced FliE-V99G was not negatively dominant over wild-type FliE. We conclude that the mutant protein has reduced probability of assembly but, if assembled, functions relatively well. Export of the flagellar protein FlgD, which is known to be FliE dependent, was severely impaired by the FliE-V99G mutation but was significantly improved in the suppressor strains. The FliE mutation, V99G, was close to the C terminus of the 104-amino-acid sequence; the suppressing mutations in FlgB were all either G119E or G129D, close to the C terminus of its 138-amino-acid sequence. Affinity blotting experiments between FliE as probe and various basal body proteins as targets and vice versa revealed strong interactions between FliE and FlgB; much weaker interactions between FliE and other rod proteins were observed and probably derive from the known similarities among these proteins. We suggest that FliE subunits constitute a junction zone between the MS ring and the rod and also that the proximal rod structure consists of FlgB subunits.     

In vitro mutagenesis of the mitochondrial leucyl tRNA synthetase ofSaccharomyces cerevisiae shows that the suppressor activity of the mutant proteins is related to the splicing function of the wild-type protein

TheNAM2 gene ofSaccharomyces cerevisiae encodes the mitochondrial leucyl tRNA synthetase (mLRS), which is necessary for the excision of the fourth intron of the mitochondrialcytb gene (bI4) and the fourth intron of the mitochondrialcoxI gene (aI4), as well as for mitochondrial protein synthesis. Some dominant mutant alleles of the gene are able to suppress mutations that inactivate the bI4 maturase, which is essential for the excision of the introns aI4 and bI4. Here we report mutagenesis studies which focus on the splicing and suppressor functions of the protein. Small deletions in the C-terminal region of the protein preferentially reduce the splicing, but not the synthetase activity; and all the C-terminal deletions tested abolish the suppressor activity. Mutations which increase the volume of the residue at position 240 in the wild-type mLRS without introducing a charge, lead to a suppressor activity. The mutant 238C, which is located in the suppressor region, has a reduced synthetase activity and no detectable splicing activity. These data show that the splicing and suppressor functions are linked and that the suppressor activity of the mutant alleles results from a modification of the wild-type splicing activity.     

In vitro mutagenesis of the mitochondrial leucyl tRNA synthetase ofSaccharomyces cerevisiae shows that the suppressor activity of the mutant proteins is related to the splicing function of the wild-type protein

TheNAM2 gene ofSaccharomyces cerevisiae encodes the mitochondrial leucyl tRNA synthetase (mLRS), which is necessary for the excision of the fourth intron of the mitochondrialcytb gene (bI4) and the fourth intron of the mitochondrialcoxI gene (aI4), as well as for mitochondrial protein synthesis. Some dominant mutant alleles of the gene are able to suppress mutations that inactivate the bI4 maturase, which is essential for the excision of the introns aI4 and bI4. Here we report mutagenesis studies which focus on the splicing and suppressor functions of the protein. Small deletions in the C-terminal region of the protein preferentially reduce the splicing, but not the synthetase activity; and all the C-terminal deletions tested abolish the suppressor activity. Mutations which increase the volume of the residue at position 240 in the wild-type mLRS without introducing a charge, lead to a suppressor activity. The mutant 238C, which is located in the suppressor region, has a reduced synthetase activity and no detectable splicing activity. These data show that the splicing and suppressor functions are linked and that the suppressor activity of the mutant alleles results from a modification of the wild-type splicing activity.     

Loss of outer membrane proteins without inhibition of lipid export in an Escherichia coli YaeT mutant

Escherichia coli yaeT encodes an essential, conserved outer membrane (OM) protein that is an ortholog of Neisseria meningitidis Omp85. Conflicting data with N. meningitidis indicate that Omp85 functions either in assembly of OM proteins or in export of OM lipids. The role of YaeT in E. coli was investigated with a new temperature-sensitive mutant harboring nine amino acid substitutions. The mutant stops growing after 60 min at 44 degrees C. After 30 min at 44 degrees C, incorporation of [35S]methionine into newly synthesized OM proteins is selectively inhibited. Synthesis and export of OM phospholipids and lipopolysaccharide are not impaired. OM protein levels are low, even at 30 degrees C, and the buoyant density of the OM is correspondingly lower. By Western blotting, we show that levels of the major OM protein OmpA are lower in the mutant in whole cells, membranes, and the growth medium. SecA functions as a multicopy suppressor of the temperature-sensitive phenotype and partially restores OM proteins. Our data are consistent with a critical role for YaeT in OM protein assembly in E. coli.     

Mutagenesis of hetR reveals amino acids necessary for HetR function in the heterocystous cyanobacterium Anabaena sp. strain PCC 7120

HetR is the master regulator of heterocyst differentiation in the filamentous cyanobacterium Anabaena sp. strain PCC 7120. Genetic selection was used to identify 33 amino acid substitutions in HetR that reduced the proportion of cells undergoing heterocyst differentiation to less than 2%. Conservative substitutions in the wild-type HetR protein revealed three mutations that dramatically reduced the amount of heterocyst differentiation when the mutant allele was present in place of the wild-type allele on a replicating plasmid in a mutant lacking hetR on the chromosome. An H69Y substitution resulted in heterocyst formation among less than 0.1% of cells, and D17E and G36A substitutions resulted in a Het- phenotype, compared to heterocyst formation among approximately 25% of cells with the wild-type hetR under the same conditions. The D17E substitution prevented DNA binding activity exhibited by wild-type HetR in mobility shift assays, whereas G36A and H69Y substitutions had no affect on DNA binding. D17E, G36A, and H69Y substitutions also resulted in higher levels of the corresponding HetR protein than of the wild-type protein when each was expressed from an inducible promoter in a hetR deletion strain, suggesting an effect on HetR protein turnover. Surprisingly, C48A and S152A substitutions, which were previously reported to result in a Het- phenotype, were found to have no effect on heterocyst differentiation or patterning when the corresponding mutations were introduced into an otherwise wild-type genetic background in Anabaena sp. strain PCC 7120. The clustering of mutations that satisfied the positive selection near the amino terminus suggests an important role for this part of the protein in HetR function.     

Thermo-labile stability of sigmaH (Spo0H) in temperature-sensitive spo0H mutants of Bacillus subtilis can be suppressed by mutations in RNA polymerase beta subunit

We isolated novel temperature-sensitive mutants of spo0H, spo0H1 and spo0H5, having E61K and G30E amino-acid substitutions within the sigmaH protein, respectively, and located in the highly conserved region, "2", among prokaryotic sigma factors that participates in binding to core enzyme of RNA polymerase. These mutants showed a sporulation-deficient phenotype at 43 degrees C. Moreover, we successfully isolated suppressor mutants that were spontaneously generated from the spo0H mutants. Our genetic analysis of these suppressor mutations revealed that the suppressor mutations are within the rpoB gene coding for the beta subunit of RNA polymerase. The mutations caused single amino-acid substitutions, E857A and P1055S, in rpoB18 and rpoB532 mutants that were generated from spo0H1 and spo0H5, respectively. Whereas the sigmaH-dependent expression of a spo0A-bgaB fusion was greatly reduced in both spo0H mutants, their expression was partially restored in the suppressor mutants at 43 degrees C. Western blot analysis showed that the level of sigmaH protein in the wild type increased between T0 and T2 and decreased after T3, while the level of sigmaH protein in spo0H mutants was greatly reduced throughout growth, indicating that the mutant sigmaH proteins were rapidly degraded by some unknown proteolytic enzyme(s). The analysis of the half-life of sigmaH protein showed that the short life of sigmaH in spo0H mutants is prolonged in the suppressor mutants. These findings suggest that, at least to some extent, the process of E-sigmaH formation may be involved in stabilization of sigmaH at the onset of sporulation.     

Phage λ Cro Protein and Ci Repressor Use Two Different Patterns of Specific Protein-DNA Interactions to Achieve Sequence Specificity in Vivo

By assaying the binding of wild-type Cro to a set of 40 mutant lambda operators in vivo, we have determined that the 14 outermost base pairs of the 17 base pair, consensus lambda operator are critical for Cro binding. Cro protein recognizes 4 base pairs in a lambda operator half-site in different ways than cI repressor. The sequence determinants of Cro binding at these critical positions in vivo are nearly perfectly consistent with the model proposed by W. F. ANDERSON, D. H. OHLENDORF, Y. TAKEDA and B. W. MATTHEWS and modified by Y. TAKEDA, A. SARAI and V. M. RIVERA for the specific interactions between Cro and its operator, and explain the relative order of affinities of the six natural lambda operators for Cro. Our data call into question the idea that lambda repressor and Cro protein recognize the consensus lambda operator by nearly identical patterns of specific interactions.