The antigenic surface of staphylococcal nuclease. II. Analysis of the N-1 epitope by site-directed mutagenesis.

Previous studies in our laboratory on the production and isolation of a panel of mAb to staphylococcal nuclease allowed us to define a series of eight overlapping epitopes. Using site-directed mutagenesis of the nuclease coding sequences we were able to map the nonoverlapping epitopes recognized by two members of this panel. In the study reported here, we report the generation and analysis of a number of single amino acid substitutions for seven surface residues predicted to lie within one of these two epitopes. Immunochemical analysis showed that one or more substitutions at each of these seven positions had a major effect on mAb binding, whereas other substitutions had none. Based on the nature of these substitutions and the chemical and physical properties of the variant molecules, we believe that any structural effects induced by these substitutions are local and do not result in long-range structural alterations that indirectly influence antibody reactivity. Therefore, we conclude that disruption of mAb binding can be directly attributed to changes in amino acid side chains and that not only are all seven of the residues studied part of the epitope but all seven make contact with the antibody combining site. These studies demonstrate the advantages of using site-directed mutagenesis to study antigen structure and emphasize the importance of constructing the examining multiple substitutions for any given amino acid.     

Analysis of the catalytic mechanism of juvenile hormone esterase by site-directed mutagenesis.

1. Juvenile hormone esterase (JHE) is a serine hydrolase selective for hydrolysis of the conjugated methyl esters of insect juvenile hormones. 2. We have investigated the mechanism of catalytic action of this enzyme by site-directed mutagenesis of the cloned enzyme and expression of the mutants in a baculovirus system. 3. A series of individual mutations of JHE were made to residues possibly involved in catalysis of juvenile hormones, and which are highly conserved in both esterases and lipases. 4. Mutation of the serine residue at position 201 to glycine (S201G), or aspartate 173 to asparagine (D173N), or histidine 446 to lysine (H446K), removed all detectable activity and these mutagenized enzymes were determined to be at least 10(6)-fold less active than wild type JHE. 5. Mutation of arginine 47 to histidine (R47H) decreased but did not abolish activity, with Km essentially unchanged at 66 nM for R47H compared to 34 nM for wild type JHE. 6. The kcat for R47H was decreased from 103 min-1 for wild type JHE to 1.9 min-1. 7. In addition, glutamate residue 332 was altered to glutamine (E332Q) and expressed in an Escherichia coli system. 8. This mutation was also found to remove all detectable activity. 9. From the results presented in this study and by comparison of JHE to other serine esterases and lipases, we predict that JHE possesses a Ser201-His446-Glu332 catalytic triad. 10. In addition, aspartate 173 and arginine 47 are essential for the efficient functioning of JHE.     

The antigenic surface of staphylococcal nuclease. I. Mapping epitopes by site-directed mutagenesis.

The analysis of the antigenic surface of staphylococcal nuclease was begun by generating and characterizing a panel of mAb. Twelve mAb were selected from a large number of anti-nuclease mAb and characterized for affinity and isotype, by their ability to block enzyme activity, and by complementation and competitive inhibition assays for the relative location of epitopes. The mAb were placed in complementation groups based on their distinct binding patterns. These groups define a series of eight overlapping epitopes that are estimated to cover a large portion of the nuclease surface. Four mAb blocked the enzyme activity of nuclease. The epitopes defined by two of these four mAb were localized on the surface of nuclease using single amino acid variant Ag generated by site-directed mutagenesis of the cloned nuclease coding sequence. mAb-25 maps to residue 46 which is located at the edge of the enzyme active site consistent with its ability to inhibit enzyme activity. mAb-19, which also blocks enzyme activity and belongs to the same complementation group as mAb-25, was unaffected by the substitution at position 46. This suggests that mAb-19 and mAb-25, if they do react with the same epitope, have differences in fine specificity. mAb-22 blocks enzyme activity and belongs to an overlapping complementation group. The fourth mAb, mAb-1, which belongs to a distinct, nonoverlapping, complementation group, does not blocks enzyme activity, and is directed to a region of nuclease that includes the amino acid at position 133. This residue is located a short distance from the active site in a region that has been suggested to participate in binding of DNA, a substrate for nuclease. Therefore, the four epitopes defined by these mAb are localized at or near the enzyme active site.     

Analysis of the ends of bacteriophage Mu using site-directed mutagenesis

We showed previously that two regions at the left end (L1 and L3) and one at the right end (R2) of bacteriophage Mu are essential for transposition. These regions all contain a 22 base-pair sequence with the consensus YGtTTCAYtNNAARYRCGAAAR, where Y and R represent any pyrimidine and purine, respectively. The Mu A protein binds to these regions in vitro, and weakly to sequences between nucleotides 1 and 30 of the right end (R1) and between nucleotides 110 and 135 of the left end (L2). These weak A binding sites contain the sequence AARYRCGAAAR. Here we show, using site-directed mutagenesis, that the weak A binding sites are essential for transposition. Mutations in these weak A binding sites have a greater effect on transposition than mutations of corresponding base-pairs in the stronger A binding sites, located adjacent to these weak A binding sites. We confirm the importance of several of the conserved base-pairs in the consensus sequence YGtTTCAYtNNAARYRCGAAAR. The base-pairs in the A binding sites that are shown to be essential for transposition are all conserved in the ends of the related bacteriophage D108. Furthermore, it is shown that the distance of 90 base-pairs between the two regions at the left end (L1 and L2L3) is essential.     

In vivo analysis of the Saccharomyces cerevisiae HO nuclease recognition site by site-directed mutagenesis.

HO nuclease introduces a specific double-strand break in the mating-type locus (MAT) of Saccharomyces cerevisiae, initiating mating-type interconversion. To define the sequence recognized by HO nuclease, random mutations were produced in a 30-base-pair region homologous to either MAT alpha or MATa by a chemical synthesis procedure. The mutant sites were introduced into S. cerevisiae on a shuttle vector and tested for the ability to stimulate recombination in an assay that mimics mating-type interconversion. The results suggest that a core of 8 noncontiguous bases near the Y-Z junction of MAT is essential for HO nuclease to bind and cleave its recognition site. Other contacts must be required because substrates that contain several mutations outside an intact core reduce or eliminate cleavage in vivo. The results show that HO site recognition is a complex phenomenon, similar to promoter-polymerase interactions.     

Efficient site-directed saturation mutagenesis using degenerate oligonucleotides.

We describe a reliable protocol for constructing single-site saturation mutagenesis libraries consisting of all 20 naturally occurring amino acids at a specific site within a protein. Such libraries are useful for structure-function studies and directed evolution. This protocol extends the utility of Stratagene's QuikChange Site-Directed Mutagenesis Kit, which is primarily recommended for single amino acid substitutions. Two complementary primers are synthesized, containing a degenerate mixture of the four bases at the three positions of the selected codon. These primers are added to starting plasmid template and thermal cycled to produce mutant DNA molecules, which are subsequently transformed into competent bacteria. The protocol does not require purification of mutagenic oligonucleotides or PCR products. This reduces both the cost and turnaround time in high-throughput directed evolution applications. We have utilized this protocol to generate over 200 site-saturation libraries in a DNA polymerase, with a success rate of greater than 95%.     

Significance of local electrostatic interactions in staphylococcal nuclease studied by site-directed mutagenesis

In this paper, we show that amino acids Glu(73) and Asp(77) of staphylococcal nuclease cooperate unequally with Glu(75) to stabilize its structure located between the C-terminal helix and beta-barrel of the protein. Amino acid substitutions E73G and D77G cause losses of the catalytic efficiency of 24 and 16% and cause thermal stability losses of 22 and 26%, respectively, in comparison with the wild type (WT) protein. However, these changes do not significantly change global and local secondary structures, based on measurements of fluorescence and CD(222 nm). Furthermore, x-ray diffraction analysis of the E75G protein shows that the overall structure of mutant and WT proteins is similar. However, this mutation does cause a loss of essential hydrogen bonding and charge interactions between Glu(75) and Lys(9), Tyr(93), and His(121). In experiments using double point mutations, E73G/D77G, E73G/E75G, and E75G/D77G, significant changes are seen in all mutants in comparison with WT protein as measured by fluorescence and CD spectroscopy. The losses of thermal stability are 47, 59, and 58%, for E73G/D77G, E73G/E75G, and E75G/D77G, respectively. The triple mutant, E73G/E75G/D77G, results in fluorescence intensity and CD(222 nm) close to those of the denatured state and in a thermal stability loss of 65% relative to the WT protein. Based on these results, we propose a model in which significant electrostatic interactions result in the formation of a locally stable structure in staphylococcal nuclease.     

Role of serine 352 in the allosteric response of Serratia marcescens aspartokinase I-homoserine dehydrogenase I analyzed by using site-directed mutagenesis.

Aspartokinase I and homoserine dehydrogenase I (AKI-HDI) from Serratia marcescens Sr41 are encoded by the thrA gene as a single polypeptide chain. Previously, a single amino acid substitution of Ser-352 with Phe was shown to produce an AKI-HDI enzyme that is not subject to threonine-mediated feedback inhibition. To determine the role of Ser-352 in the allosteric response, the thrA gene was modified by using site-directed mutagenesis so that Ser-352 of the wild-type AKI-HDI was replaced by Ala, Arg, Asn, Gln, Glu, His, Leu, Met, Pro, Thr, Trp, Tyr, or Val. The Thr-352 and Pro-352 replacements rendered AKIs sensitive to threonine. The Tyr-352 and Asn-352 substitutions led to activation, rather than inhibition, of AKI by threonine. The other replacements conferred threonine insensitivity on AKI. The threonine sensitivity of HDI was also changed by the amino acid substitutions at Ser-352. The HDI carried by the Tyr-352 mutant AKI-HDI was activated by threonine. Single amino acid replacements at Ser-352 by Ala, Asn, Gln, His, Phe, Pro, Thr, or Tyr were introduced into truncated AKI-HDIs containing the AKI and the central regions. The AKI activity of the truncated AKI-HDI containing the first 468 amino acid residues was sensitive to threonine, and introduction of the amino acid replacements did not alter the threonine sensitivity of the AKI. Another truncated AKI-HDI containing the first 462 amino acid residues possessed threonine-resistant AKI, whereas the substitutions of Ser-352 with Ala and Pro rendered AKI sensitive to threonine. The replacement of GIn-351 with Phe activated AK1 of the truncated AKI-HDI in the presence of L-threonine. These findings suggest that Ser-352 of the central region of AKI-HDI is possibly a key residue involved with the allosteric regulation of both AKI and HDI activities.     

Improved oligonucleotide site-directed mutagenesis using M13 vectors.

An improved method is described for the construction of mutations in M13 vectors using synthetic oligonucleotides. The DNA is first cloned into a novel M13 vector (based upon M13mp18 or M13mp19), which carries a genetic marker that can be selected against, such as an EcoK or EcoB site, or an amber mutation in an essential phage gene. In this "coupled priming" technique, one primer is used to construct the silent mutation of interest, and a second primer is used to eliminate the selectable marker on the minus strand. After primer extension and ligation, the heteroduplex DNA is transfected into a strain of E. coli which is repair deficient and selects against the plus strand marker. Over 50 mutants have been constructed with this approach, and the yields can be excellent (up to 70%). For the stepwise construction of mutations using separate rounds of mutagenesis, the EcoK and EcoB markers offer a particular advantage over the amber marker. They permit selection in each round, as it is possible to cycle between the two markers. However for construction of multiple mutations over a short region, long synthetic oligonucleotides with multiple mismatches to the template can offer an alternative strategy.     

In vivo site-directed mutagenesis using oligonucleotides

Functional characterization of the genes of higher eukaryotes has been aided by their expression in model organisms and by analyzing site-specific changes in homologous genes in model systems such as the yeast Saccharomyces cerevisiae. Modifying sequences in yeast or other organisms such that no heterologous material is retained requires in vitro mutagenesis together with subcloning. PCR-based procedures that do not involve cloning are inefficient or require multistep reactions that increase the risk of additional mutations. An alternative approach, demonstrated in yeast, relies on transformation with an oligonucleotide, but the method is restricted to the generation of mutants with a selectable phenotype. Oligonucleotides, when combined with gap repair, have also been used to modify plasmids in yeast; however, this approach is limited by restriction-site availability. We have developed a mutagenesis approach in yeast based on transformation by unpurified oligonucleotides that allows the rapid creation of site-specific DNA mutations in vivo. A two-step, cloning-free process, referred to as delitto perfetto, generates products having only the desired mutation, such as a single or multiple base change, an insertion, a small or a large deletion, or even random mutations. The system provides for multiple rounds of mutation in a window up to 200 base pairs. The process is RAD52 dependent, is not constrained by the distribution of naturally occurring restriction sites, and requires minimal DNA sequencing. Because yeast is commonly used for random and selective cloning of genomic DNA from higher eukaryotes such as yeast artificial chromosomes, the delitto perfetto strategy also provides an efficient way to create precise changes in mammalian or other DNA sequences.     

Analysis of the diphtheria tox promoter by site-directed mutagenesis.

By oligonucleotide-directed mutagenesis, we introduced alterations in the two putative -10 regions of the diphtheria tox promoter which are positioned at -50 and -56 from the GUG tox initiation signal. The -10 region positioned at -50 is favored in the expression of ADP-ribosyltransferase activity from the wild-type tox promoter in recombinant Escherichia coli; however, the promoter down mutation at position -50 is compensated for by increased activity of the -10 region positioned at -56.     

Evaluation of the mechanism of aromatase cytochrome P450. A site-directed mutagenesis study.

Aromatase (CYP19) catalyzes three consecutive hydroxylation reactions converting C19 androgens to aromatic C18 estrogenic steroids. In this study, five human aromatase mutants (E302D, S478A, S478T, H480K, and H480Q) were prepared using a mammalian cell expression system. These mutants were evaluated by enzyme kinetic analysis, inhibitory profile studies, and reaction intermediate measurements. Three steroidal inhibitors [4-hydroxyandrostenedione (4-OHA), 7alpha-(4'-amino)phenylthio-1,4-androstandiene-3,17-dione (7alpha-APTADD), and bridge (2,19-methyleneoxy) androstene-3,17-dione (MDL 101003)], and four nonsteroidal inhibitors [aminoglutethimide (AG), CGS 20267, ICI D1033, and vorozole (R83842)] were used in the inhibitory profile studies. Our computer model of aromatase suggests that Glu302 is situated in the conserved I-helix region and located near the C-19 position of the steroid substrate. The model was supported by significant changes in kinetic parameters and a sevenfold increase in the Ki value of MDL 101,003 for the mutant E302D. As S478A was found to have kinetic properties similar to the wild-type enzyme and a much higher activity than S478T, Ser478 is thought to be situated in a rather restricted environment. There was a 10-fold increase in the Ki value of 7alpha-APTADD for S478T over that for the wild-type enzyme, suggesting that Ser478 might be near the C-7 position of the substrate. The reaction intermediate analysis revealed that significantly more 19-ol intermediate was generated by both S478A and S478T than the wild-type enzyme. These results would support a hypothesis that Ser478 plays a role in the first and second hydroxylation reactions. A positive charged amino acid is preferred at position 480 as shown by the fact that H480K has a significantly higher activity than H480Q. The Ki value of 4-OHA for H480Q was found to be three times that of the wild-type enzyme. In addition, significantly more 19-ol and 19-al intermediates were detected for both mutants H480K and H480Q than for the wild-type enzyme. Evaluation of the two mutations at His480 allows us to propose that this residue may participate in the aromatization reaction (the third step) by acting as a hydrogen bond donor for the C-3 keto group of the substrate. Furthermore, new products were generated when the enzyme was mutated at Ser478 and His480. Thus, these two residues must play an important role in the catalysis and are likely closer to the substrate binding site than previously predicted.     

Substrate specificity of prorenin converting enzyme of mouse submandibular gland. Analysis using site-directed mutagenesis

Renin is produced from a larger, inactive precursor, prorenin, through endoproteolytic cleavage at paired basic amino acids. Recently, we have purified and characterized an enzyme, which catalyzes the endoproteolytic process, from mouse submandibular gland. The enzyme, named prorenin converting enzyme, specifically cleaves the peptide bond on the COOH-side of the Arg residue at the Lys-Arg pair of mouse Ren 2 prorenin, but does not cleave mouse Ren 1 and human prorenins. In this study, by synthesizing a series of mutant mouse prorenins using site-directed mutagenesis and the Xenopus oocyte expression system, we have investigated the role of the basic pair as the recognition signal for the enzyme as well as the determinant of the substrate specificity. The results indicate that the basic amino acid at the COOH-side but not at the NH2-side of the basic pair of Ren 2 prorenin is essential for processing directed by prorenin converting enzyme, and that the Arg residue at the COOH-side is more preferable for processing than the Lys. The results also demonstrated that the presence of a Pro residue next to the Lys-Arg pair prevents the processing of Ren 1 prorenin.     

A simple method for site-directed mutagenesis using the polymerase chain reaction.

We have developed a general and simple method for directing specific sequence changes in a plasmid using primed amplification by the polymerase chain reaction (PCR). The method is based on the amplification of the entire plasmid using primers that include the desired changes. The method is rapid, simple in its execution, and requires only minute amounts of plasmid template DNA. It is significant that there are no special requirements for appropriately placed restriction sites in the sequence to be manipulated. In our system the yield of transformants was high and the fraction of them harboring plasmids with only the desired change was consistently about 80%. The generality of the method should make it useful for the direct alteration of most cloned genes. The only limitation may be the total length of the plasmid to be manipulated. During the study we found that the Taq DNA polymerase used for PCR adds on a single extra base (usually an A) at the end of a large fraction of the newly synthesized chains. These had to be removed by the Klenow fragment of DNA polymerase to insure restoration of the gene sequence.     

Overexpression, site-directed mutagenesis, and mechanism of Escherichia coli acid phosphatase.

Site-directed mutagenesis was used to examine the catalytic importance of 2 histidine and 4 arginine residues in Escherichia coli periplasmic acid phosphatase (EcAP). The residues that were selected as targets for mutagenesis were those that were also conserved in a number of high molecular weight acid phosphatases from eukaryotic organisms, including human prostatic and lysosomal acid phosphatases. Both wild type EcAP and mutant proteins were overproduced in E. coli using an expression system based on the T7 RNA polymerase promoter, and the proteins were purified to homogeneity. Examination of the purified mutant proteins by circular dichroism and proton NMR spectroscopy revealed no significant conformational changes. The replacement of Arg16 and His17 residues that were localized in a conserved N-terminal RHGXRXP motif resulted in the complete elimination of EcAP enzymatic activity. Critical roles for Arg20, Arg92, and His303 were also established because the corresponding mutant proteins exhibited residual activities that were not higher than 0.4% of that of wild type enzyme. In contrast, the replacement of Arg63 did not cause a significant alteration of the kinetic parameters. The results are in agreement with a previously postulated distant relationship between acid phosphatases, phosphoglycerate mutases, and fructose-2,6-bisphosphatase. These and earlier results are also consistent with the conclusion that 2 histidine residues participate in the catalytic mechanism of acid phosphatases, with His17 playing the role of a nucleophilic acceptor of the phospho group, whereas His303 may act as a proton donor to the alcohol or phenol.     

Efficient site-directed in vitro mutagenesis using phagemid vectors

Several methods have been developed that enhance the efficiency of in vitro, site-directed mutagenesis. Kunkel (8,9) has developed a method which uses a strong selection for the mutated strand and, hence, is highly efficient, but yet simple and rapid. This method originally used M13 phage as the vector. In this paper, we describe a refinement of this method using phagemid vectors, which combine the advantages of plasmids (such as high copy number and stability of cloned DNA) with the single-stranded DNA generating capability of M13 phage. We demonstrate that high efficiency of mutant production can be obtained with these vectors. We also analyzed by sequencing 11 mutated clones and found no second-site mutations, suggesting that alterations other than the site-directed mutation rarely occur in our system.