Conformational effects of amino acid substitutions at positions 10, 12, and 13 in the P21 protein

Substitutions of amino acids for Gly 12 or Gly 13 in theras oncogene-encoded P21 proteins have been demonstrated to produce unique structural changes in these proteins that correlate with their ability to produce cell transformation. For example, the P21 proteins with Arg 12 or Val 13 are both known to be actively transforming. Recent site-specific mutagenesis experiments on the transforming Arg 12 protein have found that the substitution of Val for Gly 10 has no effect on transforming activity whereas the substitution of Val for Gly 13 led to a loss of transforming activity. In this study, we examine the structural effects of these substitutions on the amino terminal hydrophobic decapeptide (Leu 6-Gly 15) of P21 using conformational energy analysis. The results show that the transforming proteins with Gly 10 and Arg 12 or Val 10 and Arg 12 can both adopt the putative malignancy-causing conformation, whereas, for the nontransforming protein with Arg 12 and Val 13, this conformation is energetically disallowed. These results further support the theory that due to structural changes the transforming P21 proteins are unable to bind to some regulatory cellular element which may be the recently identified binding protein responsible for the induction of increased GTPase activity in normal P21 compared with transforming mutants.     

Structural effects of amino acid substitutions on the P21 proteins: Evidence for a malignant conformation

The conformational effects of different amino acid substitutions for Gly at position 12 in theras-oncogene-encoded P21 proteins have been investigated using conformational energy calculations. Mutations that cause amino acid substitutions for Gly 12 result in a protein that produces malignant transformation of cells. It had previously been shown that substitution of Val, Lys, or Ser for Gly at position 12 results in a major conformational change, and that the preferred lowest energy structure for each of the substituted peptides is identical. It is now found that substitution for Gly 12 of other amino acids that have widely disparate helix-nucleating potentials and completely different side chains (Asp, Asn, Cys, Phe, Tle, Leu, and Ala) all produce this identical lowest energy conformation. This finding is consistent with the recent results of site-specific mutagenesis experiments showing that P21 proteins containing these amino acids at position 12 all promote malignant transformation of cells and suggests the existence of a malignancy-causing conformation for the P21 proteins.     

Conformational effects of the substitution of Arg for Gly 13 in the ras oncogene-encoded P21 protein

The effect of the substitution of Arg for Gly 13 on the structure of the transforming region decapeptide (Leu 6-Gly 15) of the ras oncogene encoded P21 protein has been investigated using conformational energy analysis. A human malignancy has been identified that contains a ras gene with a single mutation in the thirteenth codon such that the encoded protein would have Arg substituted for Gly at this position, and transfection of cells in culture with this gene results in malignant transformation. Conformational analysis demonstrates that the Arg 13 decapeptide adopts a conformation identical to that for other peptides with substitutions at position 13 (Asp 13, Val 13) from transforming proteins that is distinctively different from that for peptides (Gly 13, Ser 13) from normal, nontransforming proteins. This is found to be an indirect effect resulting from changes in the conformation of Gly 12 produced by substitutions at position 13. These results are consistent with recent analysis of crystallographic data of proteins on conformational preferences for glycine in tripeptide sequences.     

Correlation of structure with transforming activity of the P21 proteins with substitutions of L-amino acids for Gly at position 13

The effects of amino acid substitutions for Gly 13 on the structure of the transforming region (Leu 6-Gly 15) of the P21 proteins have been explored using conformational energy calculations. It has been found that the substitution of Asp for Gly at this position results in a protein capable of transforming cells into malignant ones. Proteins that contain Ser at position 13 (but no other substitutions), however, transform cells with a greatly reduced activity. The transforming peptide with Asp 13 adopts a conformation that is different from the one for the peptide from the normal protein (with Gly 12 and Gly 13) and that may result in expression of a higher energy malignancy-producing form. The Ser-containing peptide adopts as its lowest energy conformation one that is identical to that of the peptide from the normal protein, thus explaining its lack of transforming activity. From analysis of the interactions preventing the Asp 13-containing peptide from adopting the normal conformation, it is predicted that substitutions of amino acids with branched side chains atC , such as Val, Ile, and Thr, should promote cell transformation. This prediction with Val has recently been confirmed in genetic experiments.     

Comparison of the computed structures for the phosphate-binding loop of the p21 protein containing the oncogenic site Gly 12 with the X-ray crystallographic structures for this region in the p21 protein and EFtu. A model for the structure of the p21 protein in its oncogenic form

The GTP-binding p21 protein encoded by the ras-oncogene can be activated to cause malignant transformation of cells by substitution of a single amino acid at critical positions along the polypeptide chain. Substitution of any non-cyclic L-amino acid for Gly 12 in the normal protein results in a transforming protein. This substitution occurs in a hydrophobic sequence (residues 6-15) which is known to be involved in binding the phosphate moities of GTP (and GDP). We find, using conformational energy calculations, that the 6-15 segment of the normal protein (with Gly 12) adopts structures that contain a bend at residues 11 and 12 with the Gly in the D* conformation, not allowed energetically for L-amino acids. Substitution of non-cyclic L-amino acids for Gly 12 results in shifting this bend to residues 12 and 13. We show that many computed structures for the Gly 12-containing phosphate binding loop, segment 9-15, are superimposable on the corresponding segment of the recently determined X-ray crystallographic structure for residues 1-171 of the p21 protein. All such structures contain bends at residues 11 and 12 and most of these contain Gly 12 in the C* or D* conformational state. Other computed conformations for the 9-15 segment were superimposable on the structure of the corresponding 18-23 segment of EFtu, the bacterial chain elongation factor having structural similarities to the p21 protein in the phosphate-binding regions. This segment contains a Val residue where a Gly occurs in the p21 protein. As previously predicted, all of these superimposable conformations contain a bend at positions 12 and 13, not 11 and 12. If these structures that are superimposable on EFtu are introduced into the p21 protein structure, bad contacts occur between the sidechain of the residue (here Val) at position 12 and another phosphate binding loop region around position 61. These bad contacts between the two segments can be removed by changing the conformation of the 61 region in the p21 protein to the corresponding position of the homologous region in EFtu. In this new conformation, a large site becomes available for the binding of phosphate residues. In addition, such phenomena as autophosphorylation of the p21 protein by GTP can be explained with this new model structure for the activated protein which cannot be explained by the structure for the non-activated protein.     

RSR1, a ras-like gene homologous to Krev-1 (smg21A/rap1A): role in the development of cell polarity and interactions with the Ras pathway in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae ras-like gene RSR1 is particularly closely related to the mammalian gene Krev-1 (also known as smg21A and rap1A). RSR1 was originally isolated as a multicopy suppressor of a cdc24 mutation, which causes an inability to bud or establish cell polarity. Deletion of RSR1 itself does not affect growth but causes a randomization of bud position. We have now constructed mutant alleles of RSR1 encoding proteins with substitutions of Val for Gly at position 12 (analogous to constitutively activated Ras proteins) or Asn for Lys at position 16 (analogous to a dominant-negative Ras protein). rsr1Val-12 could not restore a normal budding pattern to an rsr1 deletion strain but could suppress a cdc24 mutation when overexpressed. rsr1Asn-16 could randomize the budding pattern of a wild-type strain even in low copy number but was not lethal even in high copy number. These and other results suggest that Rsr1p functions only in bud site selection and not in subsequent events of polarity establishment and bud formation, that this function involves a cycling between GTP-bound and GDP-bound forms of the protein, and that the suppression of cdc24 involves direct interaction between Rsr1p[GTP] and Cdc24p. Functional homology between Rsr1p and Krev-1 p21 was suggested by the observations that expression of the latter protein in yeast cells could both suppress a cdc24 mutation and randomize the budding pattern of wild-type cells. As Krev-1 overexpression can suppress ras-induced transformation of mammalian cells, we looked for effects of RSR1 on the S. cerevisiae Ras pathway. Although no suppression of the activated RAS2Val-19 allele was observed, overexpression of rsr1Val-12 suppressed the lethality of strains lacking RAS gene function, apparently through a direct activation of adenyl cyclase. This interaction of Rsr1p with the effector of Ras in S. cerevisiae suggests that Krev-1 may revert ras-induced transformation of mammalian cells by affecting the interaction of ras p21 with its effector.     

Conformational effects of the substitution of ARG for GLY 13 in theras oncogene-encoded P21 protein

The effect of the substitution of Arg for Gly 13 on the structure of the transforming region decapeptide (Leu 6-Gly 15) of the ras oncogene encoded P21 protein has been investigated using conformational energy analysis. A human malignancy has been identified that contains a ras gene with a single mutation in the thirteenth codon such that the encoded protein would have Arg substituted for Gly at this position, and transfection of cells in culture with this gene results in malignant transformation. Conformational analysis demonstrates that the Arg 13 decapeptide adopts a conformation identical to that for other peptides with substitutions at position 13 (Asp 13, Val 13) from transforming proteins that is distinctively different from that for peptides (Gly 13, Ser 13) from normal, nontransforming proteins. This is found to be an indirect effect resulting from changes in the conformation of Gly 12 produced by substitutions at position 13. These results are consistent with recent analysis of crystallographic data of proteins on conformational preferences for glycine in tripeptide sequences.     

Effect of amino acid residues on conformational stability in eight mutant proteins variously substituted at a unique position of the tryptophan synthase alpha-subunit

To elucidate the role of individual amino acid residues in stabilizing the conformation of a protein, the stabilities of wild-type tryptophan synthase alpha-subunit from Escherichia coli and seven mutant proteins substituted by single amino acid residues at position 49, which is buried in the interior of the protein, were compared. The mutant proteins have Gln, Met, Val, Tyr, Leu, Ser, or Lys in place of Glu in the wild-type protein. The dissociation constant, pK, of the Glu residue at position 49 for the wild-type protein was determined to be 7.5 from a titration curve obtained by comparison of two-dimensional isoelectric focusing electrophoresis of the wild-type and mutant proteins. Our results indicate that 1) the conformational stabilities of the proteins studied increase linearly with hydrophobicity of the substituting residues (except Tyr), with the coefficient of this linear dependence being 2.0, 3.4, or 1.3 at pH 5.5, 7.0, or 9.0, respectively; and 2) Lys or Glu at position 49 serve as a destabilizing factor when ionized.     

Conformational changes induced by the transforming amino acid substitution in the transmembrane domain of theneu oncogene-encoded p185 protein

Theneu oncogene is frequently found in certain types of human carcinomas and has been shown to be activated in animal models by nitrosourea-induced mutation. The activating mutation in theneu oncogene results in the substitution of a glutamic acid for a valine at position 664 in the transmembrane domain of the encoded protein product of 185 kda (designated p185), which, on the basis of homology studies, is presumed to be a receptor for an as yet unidentified growth factor. It has been proposed that activating amino acid substitutions in this region of p185 lead to a conformational change in the protein which causes signal transduction via an increase in tyrosine kinase activity in the absence of any external signal. Using conformational energy analysis, we have determined the preferred three-dimensional structures for the transmembrane decapeptide (residues 658–667) of the p185 protein with valine and glutamic acid at the critical position 664. The results indicate that the global minimum energy conformation of the decapeptide from the normal protein with Val at position 664 is an α-helix with a sharp bend (CD* conformation at residues 664 and 665) in this region, whereas the global minimum conformation for the decapeptide from the mutant transforming protein with Glu at position 664 assumes an all α-helical configuration. Furthermore, the second highest energy conformation for the decapeptide from the normal protein is identical to the global minimum energy conformation for the decapeptide from the transforming protein, providing a possible explanation why overexpression of the normal protein also has a transforming effect. These results suggest there may be a normal and a transforming conformation for theneu-encoded p185 proteins which may explain their differences in transforming activity.     

Comparison of the computed three-dimensional structures of oncogenic forms (bound to GDP) of theras-Gene-Encoded p21 protein with the structure of the normal (non-transforming) wild-type protein

Theras-oncogene-encoded p21 protein becomes oncogenic if amino acid substitutions occur at critical positions in the polypeptide chain. The most commonly found oncogenic forms contain Val in place of Gly 12 or Leu in place of Gln 61. To determine the effects of these substitutions on the three-dimensional structure of the whole p21 protein, we have performed molecular dynamics calculations on each of these three proteins bound to GDP and magnesium ion to compute the average structures of each of the three forms. Comparisons of the computed average structures shows that both oncogenic forms with Val 12 and Leu 61 differ substantially in structure from that of the wild type (containing Gly 12 and Gln 61) in discrete regions: residues 10–16, 32–47, 55–74, 85–89, 100–110, and 119–134. All of these regions occur in exposed loops, and several of them have already been found to be involved in the cellular functioning of the p21 protein. These regions have also previously been identified as the most flexible domains of the wild-type protein and have been bound to be the same ones that differ in conformation between transforming and nontransforming p21 mutant proteins neither of which binds nucleotide. The two oncogenic forms have similar conformations in their carboxyl-terminal domains, but differ in conformation at residues 32–47 and 55–74. The former region is known to be involved in the interaction with at least three downstream effector target proteins. Thus, differences in structure between the two oncogenic proteins may reflect different relative affinities of each oncogenic protein for each of these effector targets. The latter region, 55–74, is known to be a highly mobile segment of the protein. The results strongly suggest that critical oncogenic amino acid substitutions in the p21 protein cause changes in the structures of vital domains of this protein.     

Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry.

The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.     

Comparison of the computed three-dimensional structures of oncogenic forms (bound to GDP) of theras-Gene-Encoded p21 protein with the structure of the normal (non-transforming) wild-type protein

Theras-oncogene-encoded p21 protein becomes oncogenic if amino acid substitutions occur at critical positions in the polypeptide chain. The most commonly found oncogenic forms contain Val in place of Gly 12 or Leu in place of Gln 61. To determine the effects of these substitutions on the three-dimensional structure of the whole p21 protein, we have performed molecular dynamics calculations on each of these three proteins bound to GDP and magnesium ion to compute the average structures of each of the three forms. Comparisons of the computed average structures shows that both oncogenic forms with Val 12 and Leu 61 differ substantially in structure from that of the wild type (containing Gly 12 and Gln 61) in discrete regions: residues 10–16, 32–47, 55–74, 85–89, 100–110, and 119–134. All of these regions occur in exposed loops, and several of them have already been found to be involved in the cellular functioning of the p21 protein. These regions have also previously been identified as the most flexible domains of the wild-type protein and have been bound to be the same ones that differ in conformation between transforming and nontransforming p21 mutant proteins neither of which binds nucleotide. The two oncogenic forms have similar conformations in their carboxyl-terminal domains, but differ in conformation at residues 32–47 and 55–74. The former region is known to be involved in the interaction with at least three downstream effector target proteins. Thus, differences in structure between the two oncogenic proteins may reflect different relative affinities of each oncogenic protein for each of these effector targets. The latter region, 55–74, is known to be a highly mobile segment of the protein. The results strongly suggest that critical oncogenic amino acid substitutions in the p21 protein cause changes in the structures of vital domains of this protein.     

Conformational changes induced by the transforming amino acid substitution in the transmembrane domain of the neu oncogene-encoded p185 protein

Theneu oncogene is frequently found in certain types of human carcinomas and has been shown to be activated in animal models by nitrosourea-induced mutation. The activating mutation in theneu oncogene results in the substitution of a glutamic acid for a valine at position 664 in the transmembrane domain of the encoded protein product of 185 kda (designated p185), which, on the basis of homology studies, is presumed to be a receptor for an as yet unidentified growth factor. It has been proposed that activating amino acid substitutions in this region of p185 lead to a conformational change in the protein which causes signal transduction via an increase in tyrosine kinase activity in the absence of any external signal. Using conformational energy analysis, we have determined the preferred three-dimensional structures for the transmembrane decapeptide (residues 658–667) of the p185 protein with valine and glutamic acid at the critical position 664. The results indicate that the global minimum energy conformation of the decapeptide from the normal protein with Val at position 664 is an -helix with a sharp bend (CD* conformation at residues 664 and 665) in this region, whereas the global minimum conformation for the decapeptide from the mutant transforming protein with Glu at position 664 assumes an all -helical configuration. Furthermore, the second highest energy conformation for the decapeptide from the normal protein is identical to the global minimum energy conformation for the decapeptide from the transforming protein, providing a possible explanation why overexpression of the normal protein also has a transforming effect. These results suggest there may be a normal and a transforming conformation for theneu-encoded p185 proteins which may explain their differences in transforming activity.     

Comparison of CD spectra in the aromatic region on a series of variant proteins substituted at a unique position of tryptophan synthase alpha-subunit

CD spectra in the aromatic region of a series of the mutant alpha-subunits of tryptophan synthase from Escherichia coli, substituted at position 49 buried in the interior of the molecule, were measured at pH 7.0 and 25 degrees C. These measurements were taken to gain information on conformational change produced by single amino acid substitutions. The CD spectra of the mutant proteins, substituted by Tyr or Trp residue in place of Glu residue at position 49, showed more intense positive bands due to one additional Tyr or Trp residue at position 49. The CD spectra of other mutant proteins also differed from that of the wild-type protein, despite the fact that the substituted residues at position 49 were not aromatic. Using the spectrum of the wild-type protein (Glu49) as a standard, the spectra of the other mutants were classified into three major groups. For 10 mutant proteins substituted by Ile, Ala, Leu, Met, Val, Cys, Pro, Ser, His, or Gly, their CD values of bands (due to Tyr residues) decreased in comparison with those of the wild-type protein. The mutant protein substituted by Phe also belonged to this group. These substituted amino acid residues are more hydrophobic than the original residue, Glu. In the second group, three mutant proteins were substituted by Lys, Gln, or Asn, and the CD values of tyrosyl bands increased compared to those of the wild-type proteins. These residues are polar. In the third group, the CD values of tyrosyl bands of two mutant proteins substituted by Asp or Thr were similar to those of the wild-type protein, except for one band at 276.5 nm. These results suggested that the changes in the CD spectra for the mutant proteins were affected by the hydrophobicity of the residues at position 49.     

Evaluation of essential and variable residues of nukacin ISK-1 by NNK scanning

Nukacin ISK-1, a type-A(II) lantibiotic, comprises 27 amino acids with a distinct linear N-terminal and a globular C-terminal region. To identify the positional importance or redundancy of individual residues responsible for nukacin ISK-1 antimicrobial activity, we replaced the native codons of the parent peptide with NNK triplet oligonucleotides in order to generate a bank of nukacin ISK-1 variants. The bioactivity of each peptide variant was evaluated by colony overlay assay, and hence we identified three Lys residues (Lys1, Lys2 and Lys3) that provided electrostatic interactions with the target membrane and were significantly variable. The ring structure of nukacin ISK-1 was found to be crucially important as replacing the ring-forming residues caused a complete loss of bioactivity. In addition to the ring-forming residues, Gly5, His12, Asp13, Met16, Asn17 and Gln20 residues were found to be essential for antimicrobial activity; Val6, Ile7, Val10, Phe19, Phe21, Val22, Phe23 and Thr24 were relatively variable; and Ser4, Pro8, His15 and Ser27 were extensively variable relative to their positions. We obtained two variants, Asp13Glu and Val22Ile, which exhibited a twofold higher specific activity compared with the wild-type and are the first reported type-A(II) lantibiotic mutant peptides with increased potency.     

The core protein of alphaviruses. 1. Purification of peptides and complete amino-acid sequence of Semliki Forest virus core protein

The primary structure of the core protein of Semliki Forest virus has been established by protein chemical characterization of 102 peptides, generated by digestion with trypsin, pepsin, thermolysin, and by partial acid cleavage of the protein. Besides a difference in one position, the sequence as established by these experiments is in agreement with the sequence predicted from the nucleotide sequence of the mRNA [Garoff et al. (1980) Proc. Natl Acad. Sci. USA, 77, 6376-6380]. The core protein has a blocked N terminus, consists of 267 amino acid residues, and has the following amino acid composition: Asp12, Asn9, Thr16, Ser10, Glu11, Gln15, Pro23, Gly20, Ala23, Val19, Met8, Ile11, Leu9, Tyr7, Phe6, His7, Lys37, Arg15, Trp5, Cys4, and an Mr of 29919. It contains 22.1% basic amino acids, mainly lysines, compared with a total of 8.6% acidic residues. The resulting surplus of positive charge is located in the N-terminal half of the protein (predominantly arginines at positions 12-21 and lysines at positions 66-114). Other amino acids are also unevenly distributed; proline and glutamine are accumulated in the N-terminal half of the sequence whereas histidine, glycine and the acidic residues are mainly present in the C-terminal part. This distribution suggests that the virus core protein consists of two or more structural domains.     

Complementation of buried lysine and surface polar residues in a designed heterodimeric coiled coil

The coiled coil is an attractive target for protein design. The helices of coiled coils are characterized by a heptad repeat of residues denoted a to g. Residues at positions a and d form the interhelical interface and are usually hydrophobic. An established strategy to confer structural uniqueness to two-stranded coiled coils is the use of buried polar Asn residues at position a, which imparts dimerization and conformational specificity at the expense of stability. Here we show that polar interactions involving buried position-a Lys residues that can interact favorably only with surface e' or g' Glu residues also impart structural uniqueness to a designed heterodimeric coiled coil with the nativelike properties of sigmoidal thermal and urea-induced unfolding transitions, slow hydrogen exchange and lack of ANS binding. The position-a Lys residues do not, however, confer a single preference for helix orientation, likely reflecting the ability of Lys at position a to from favorable interactions with g' or e' Glu residues in the parallel and antiparallel orientations, respectively. The Lys-Glu polar interaction is less destabilizing than the Asn-Asn a-->a' interaction, presumably reflecting a higher desolvation penalty associated with the completely buried polar position-a groups. Our results extend the range of approaches for two-stranded coiled-coil design and illustrate the role of complementing polar groups associated with buried and surface positions of proteins in protein folding and design.     

Conformation of the transmembrane domain of the c-erbB-2 oncogene-encoded protein in its monomeric and dimeric states

The human c-erbB-2 oncogene is homologous to the ratneu oncogene, both encoding transmembrane growth factor receptors. Overexpression and point mutations in the transmembrane domain of the encoded proteins in both cases have been implicated in cell transformation and carcinogenesis. In the case of theneu protein, it has been proposed that these effects are mediated by conformational preferences for anα-helix in the transmembrane domain, which facilitates receptor dimerization, an important step in the signal transduction process. To examine whether this is the case for c-erbB-2 as well, we have used conformational energy analysis to determine the preferred three-dimensional structures for the transmembrane domain of the c-erbB-2 protein from residues 650 to 668 with Val (nontransforming) and Glu (transforming) at position 659. The global minimum energy conformation for the Val-659 peptide from the normal, nontransforming protein was found to contain several bends, whereas the global minimum energy conformation for Glu-659 peptide from the mutant, transforming protein was found to beα-helical. Thus, the difference in conformational preferences for these transmembrane domains may explain the difference in transforming ability of these proteins. The presence of higher-energyα-helical conformations for the transmembrane domain from the normal Val-659 protein may provide an explanation for the presence of a transforming effect from overexpression of c-erbB-2. In addition, docking of the oncogenic sequences in theirα-helical and bend conformations shows that the all-α-helical dimer is clearly favored energetically over the bend dimer.     

Lysine 188 of the catabolite gene activator protein (CAP) plays no role in specificity at base pair 7 of the DNA half site.

Two similar, but not identical, models have been proposed for the amino acid-base pair contacts in the CAP-DNA complex ('model I,' Weber, I. and Steitz, T., Proc. Natl. Acad. Sci. USA, 81, 3973-3977, 1984; 'model II,' Ebright, et al., Proc. Natl. Acad. Sci. USA, 81, 7274-7278, 1984). The most important difference between the two models involves Lys188 of CAP. Model I predicts that Lys188 of CAP makes a specificity determining contact with base pair 7 of the DNA half site. In contrast, model II predicts that Lys188 makes no contact with base pair 7 of the DNA half site. In the present work, we have used site-directed mutagenesis to replace Lys188 of CAP by Asn, an amino acid unable to make the putative contact. We have assessed the specificities of the following proteins, both in vitro and in vivo: wild-type CAP, [Asn188]CAP, [Val181]CAP, and [Val181;Asn188]CAP. The results indicate that Lys188 makes no contribution to specificity at base pair 7 of the DNA half site. We propose, contrary to model I, that Lys188 makes no contact with base pair 7 of the DNA half site.     

The product of the rap2 gene, member of the ras superfamily. Biochemical characterization and site-directed mutagenesis

The human rap2 gene encodes a 183 amino acid protein that shares 46% identity with the K-ras p21. Its cDNA was engineered and inserted into the bacterial expression vector ptac; this allowed the production of high levels of soluble recombinant protein in Escherichia coli that was purified to near homogeneity. The rap2 protein binds GTP and exhibits a low intrinsic GTPase activity (rate constant of 0.5 x 10(-2) min-1). It exchanges its bound GDP with a half-life of 18 min at 37 degrees C in the presence of 10 mM Mg2+. Under the same conditions, the dissociation of bound GTP was at least 25-fold slower showing that the rap2 protein has a much higher affinity for GTP than GDP. The contribution of individual domains of the protein to its biochemical activities was investigated by site-directed mutagenesis. Substitution of Val for Gly at position 12 results in a 2-fold decrease in the GDP dissociation rate constant and GTPase activity. Replacement of the Ser at position 17 by Asn severely impairs the GTP binding ability of the protein and points to an important role of this residue in the coordination of Mg2+. Mutation of Thr-35 to Ala results in a decreased affinity for GTP and a reduction (3-fold) of the GTPase activity. Finally, substitution of Thr-145 by Ile leads to an imperfect binding of guanyl nucleotides as exemplified by an increase in their dissociation rate constants and reduction of the GTPase activity of the protein. These properties of the normal and mutant rap2 proteins are compared with those of ras p21 carrying similar substitutions and are discussed in relation to the structural models proposed for ras p21.