Constraints on amino acid substitutions in the N-terminal helix of cytochrome c explored by random mutagenesis.

The interaction of the N- and C-terminal helices is a hallmark of the cytochrome c family. Oligodeoxyribonucleotide-directed random mutagenesis within the gene encoding the C102T protein variant of Saccharomyces cerevisiae iso-1-cytochrome c was used to generate a library of mutations at the evolutionary invariant residues Gly-6 and Phe-10 in the N-terminal helix. Transformation of this library (contained on a low-copy-number yeast shuttle phagemid) into a yeast strain lacking a functional cytochrome c, followed by selection for cytochrome c function, reveals that 4-10% of the 400 possible amino acid substitutions are compatible with function. DNA sequence analysis of phagemids isolated from transformants exhibiting the functional phenotype elucidates the requirements for a stable helical interface. Basic residues are not tolerated at position 6 or 10. There is a broad volume constraint for amino acids at position 6. The amino acid substitutions observed to be compatible with function at Phe-10 show that the hydrophobic effect alone is sufficient to promote helical association. There are severe constraints that limit the combinations consistent with function, but the number of functionally consistent combinations observed exemplifies the plasticity of proteins.     

Parallel competition analysis of Saccharomyces cerevisiae strains differing by a single base using polymerase colonies

We describe a strategy to analyze the impact of single nucleotide mutations on protein function. Our method utilizes a combination of yeast functional complementation, growth competition of mutant pools and polyacrylamide gel immobilized PCR. A system was constructed in which the yeast PGK1 gene was expressed from a plasmid-borne copy of the gene in a PGK1 deletion strain of Saccharomyces cerevisiae. Using this system, we demonstrated that the enrichment or depletion of PGK1 point mutants from a mixed culture was consistent with the expected results based on the isolated growth rates of the mutants. Enrichment or depletion of individual point mutants was shown to result from increases or decreases, respectively, in the specific activities of the encoded proteins. Further, we demonstrate the ability to analyze the functional effect of many individual point mutations in parallel. By functional complementation of yeast deletions with human homologs, our technique could be readily applied to the functional analysis of single nucleotide polymorphisms in human genes of medical interest.     

Multiple lysine mutations in the C-terminal domain of p53 interfere with MDM2-dependent protein degradation and ubiquitination

To investigate the effect of mutations in the p53 C-terminal domain on MDM2-mediated degradation, we introduced single and multiple point mutations into a human p53 cDNA at four putative acetylation sites (amino acid residues 372, 373, 381, and 382). Substitution of all four lysine residues by alanines (the A4 mutant) and single lysine-to-alanine substitutions were functional in sequence-specific DNA binding and transactivation; however, the A4 mutant protein was resistant to MDM2-mediated degradation, whereas the single lysine substitutions were not. Although the A4 mutant protein and the single lysine substitutions both bound MDM2 reasonably well, the single lysine substitutions underwent normal MDM2-dependent ubiquitination, whereas the A4 protein was inefficiently ubiquitinated. In addition, the A4 mutant protein was found in the cytoplasm as well as in the nucleus of a subpopulation of cells, unlike wild-type p53, which is mostly nuclear. The partially cytoplasmic distribution of A4 mutant protein was not due to a defect in nuclear import because inhibition of nuclear export by leptomycin B resulted in nuclear accumulation of the protein. Taken together, the data suggest that mutations in the putative acetylation sites of the p53 C-terminal domain interfere with ubiquitination, thereby regulating p53 degradation.     

Human MutL homolog (MLH1) function in DNA mismatch repair: a prospective screen for missense mutations in the ATPase domain

Germline mutations in the DNA mismatch repair (MMR) genes MSH2 and MLH1 are responsible for the majority of hereditary non-polyposis colorectal cancer (HNPCC), an autosomal-dominant early-onset cancer syndrome. Genetic testing of both MSH2 and MLH1 from individuals suspected of HNPCC has revealed a considerable number of missense codons, which are difficult to classify as either pathogenic mutations or silent polymorphisms. To identify novel MLH1 missense codons that impair MMR activity, a prospective genetic screen in the yeast Saccharomyces cerevisiae was developed. The screen utilized hybrid human-yeast MLH1 genes that encode proteins having regions of the yeast ATPase domain replaced by homologous regions from the human protein. These hybrid MLH1 proteins are functional in MMR in vivo in yeast. Mutagenized MLH1 fragments of the human coding region were synthesized by error-prone PCR and cloned directly in yeast by in vivo gap repair. The resulting yeast colonies, which constitute a library of hybrid MLH1 gene variants, were initially screened by semi-quantitative in vivo MMR assays. The hybrid MLH1 genes were recovered from yeast clones that exhibited a MMR defect and sequenced to identify alterations in the mutagenized region. This investigation identified 117 missense codons that conferred a 2-fold or greater decreased efficiency of MMR in subsequent quantitative MMR assays. Notably, 10 of the identified missense codons were equivalent to codon changes previously observed in the human population and implicated in HNPCC. To investigate the effect of all possible codon alterations at single residues, a comprehensive mutational analysis of human MLH1 codons 43 (lysine-43) and 44 (serine-44) was performed. Several amino acid replacements at each residue were silent, but the majority of substitutions at lysine-43 (14/19) and serine-44 (18/19) reduced the efficiency of MMR. The assembled data identifies amino acid substitutions that disrupt MLH1 structure and/or function, and should assist the interpretation of MLH1 genetic tests.     

Random mutagenesis of Schizosaccharomyces pombe SRP RNA: lethal and conditional lesions cluster in presumptive protein binding sites.

Signal recognition particle (SRP), a ribonucleoprotein composed of six polypeptides and one RNA subunit, serves as an adaptor between the cytoplasmic protein synthetic machinery and the translocation apparatus of the endoplasmic reticulum. To begin constructing a functional map of the 7SL RNA component of SRP, we extensively mutagenized the Schizosaccharomyces pombe SRP7 gene. Phenotypes are reported for fifty-two mutant alleles derived from random point mutagenesis, seven alleles created by site-directed mutagenesis to introduce restriction sites into the SRP7 gene, nine alleles designed to pinpoint conditional lesions, and three alleles with extra nucleotides inserted at position 84. Our data indicate that virtually all single nucleotide changes as well as many multiple substitutions in this highly structured RNA are phenotypically silent. Six lethal alleles and eleven which result in sensitivity to the combination of high temperature and elevated osmotic strength were identified. These mutations cluster in conserved regions which, in the mammalian RNA, are protected from nucleolytic agents by SRP proteins. The effects of mutations in the presumptive binding site for a fission yeast SRP 9/14 homolog indicate that both the identity of a conserved residue and the secondary structure within which it is embedded are functionally important. The phenotypes of mutations in Domain IV suggest particular residues as base-specific contacts for the fission yeast SRP54 protein. A single allele which confers temperature-sensitivity in the absence of osmotic perturbants was identified in this study; the growth properties of the mutant strain suggest that the encoded RNA is somewhat defective even at the permissive temperature, and is most likely unable to correctly assemble with SRP proteins at the nonpermissive temperature.     

All-codon scanning identifies p53 cancer rescue mutations

In vitro scanning mutagenesis strategies are valuable tools to identify critical residues in proteins and to generate proteins with modified properties. We describe the fast and simple All-Codon Scanning (ACS) strategy that creates a defined gene library wherein each individual codon within a specific target region is changed into all possible codons with only a single codon change per mutagenesis product. ACS is based on a multiplexed overlapping mutagenesis primer design that saturates only the targeted gene region with single codon changes. We have used ACS to produce single amino-acid changes in small and large regions of the human tumor suppressor protein p53 to identify single amino-acid substitutions that can restore activity to inactive p53 found in human cancers. Single-tube reactions were used to saturate defined 30-nt regions with all possible codon changes. The same technique was used in 20 parallel reactions to scan the 600-bp fragment encoding the entire p53 core domain. Identification of several novel p53 cancer rescue mutations demonstrated the utility of the ACS approach. ACS is a fast, simple and versatile method, which is useful for protein structure–function analyses and protein design or evolution problems.     

Substrate range and genetic analysis of Acinetobacter vanillate demethylase

An Acinetobacter sp. genetic screen was used to probe structure-function relationships in vanillate demethylase, a two-component monooxygenase. Mutants with null, leaky, and heat-sensitive phenotypes were isolated. Missense mutations tended to be clustered in specific regions, most of which make known contributions to catalytic activity. The vanillate analogs m-anisate, m-toluate, and 4-hydroxy-3,5-dimethylbenzoate are substrates of the enzyme and weakly inhibit the metabolism of vanillate by wild-type Acinetobacter bacteria. PCR mutagenesis of vanAB, followed by selection for strains unable to metabolize vanillate, yielded mutant organisms in which vanillate metabolism is more strongly inhibited by the vanillate analogs. Thus, the procedure opens for investigation amino acid residues that may contribute to the binding of either vanillate or its chemical analogs to wild-type and mutant vanillate demethylases. Selection of phenotypic revertants following PCR mutagenesis gave an indication of the extent to which amino acid substitutions can be tolerated at specified positions. In some cases, only true reversion to the original amino acid was observed. In other examples, a range of amino acid substitutions was tolerated. In one instance, phenotypic reversion failed to produce a protein with the original wild-type sequence. In this example, constraints favoring certain nucleotide substitutions appear to be imposed at the DNA level.     

Fitness analyses of all possible point mutations for regions of genes in yeast

Deep sequencing can accurately measure the relative abundance of hundreds of mutations in a single bulk competition experiment, which can give a direct readout of the fitness of each mutant. Here we describe a protocol that we previously developed and optimized to measure the fitness effects of all possible individual codon substitutions for 10-aa regions of essential genes in yeast. Starting with a conditional strain (i.e., a temperature-sensitive strain), we describe how to efficiently generate plasmid libraries of point mutants that can then be transformed to generate libraries of yeast. The yeast libraries are competed under conditions that select for mutant function. Deep-sequencing analyses are used to determine the relative fitness of all mutants. This approach is faster and cheaper per mutant compared with analyzing individually isolated mutants. The protocol can be performed in ～4 weeks and many 10-aa regions can be analyzed in parallel.     

High-density mutagenesis by combined DNA shuffling and phage display to assign essential amino acid residues in protein-protein interactions: application to study structure-function of plasminogen activation inhibitor 1 (PAI-I)

The identification of specific amino acid residues involved in protein-protein interaction is fundamental to understanding structure-function relationships. Supported by mathematical calculations, we designed a high-density mutagenesis procedure for the generation of a mutant library of which a limited number of random clones would suffice to exactly localize amino acid residues essential for a particular protein-protein interaction. This goal was achieved experimentally by consecutive cycles of DNA shuffling, under error prone conditions, each followed by exposure of the target protein on the surface of phages to screen and select for correctly folded, functional mutants. To validate the procedure, human plasminogen activator inhibitor 1 (PAI-1) was chosen, because its 3D structure is known, many experimental tools are available and it may serve as a model protein for structure-function studies of serine proteinases and their inhibitors (serpins). After five cycles of DNA shuffling and selection for t-PA binding, analysis of 27 randomly picked clones revealed that PAI-1 mutants contained an average of 9.1 amino acid substitutions distributed over 114 different positions, which were preferentially located at the surface of the protein. This limited collection of mutant PAI-1 preparations contained multiple mutants defective in binding to three out of four tested anti-PAI-1 monoclonal antibodies. Alignment of the nucleotide sequence of defective clones permitted assignment of single dominant amino acid residues for binding to each monoclonal antibody. The importance of these residues was confirmed by testing the properties of single point mutants. From the position of these amino acid residues in the 3D structure of PAI-1 and the effects of the corresponding monoclonal antibodies on t-PA-PAI-1 interaction, conclusions can be drawn with respect to this serpin-serine proteinase interaction.     

Mutations of muscle glycogen synthase that disable activation by glucose 6-phosphate.

Glycogen synthase, an enzyme of historical importance in the field of reversible protein modification, is inactivated by phosphorylation and allosterically activated by glucose 6-phosphate (glucose-6-P). Previous analysis of yeast glycogen synthase had identified a conserved and highly basic 13-amino-acid segment in which mutation of Arg residues resulted in loss of activation by glucose-6-P. The equivalent mutations R578R579R581A (all three of the indicated Arg residues mutated to Ala) and R585R587R590A were introduced into rabbit muscle glycogen synthase. Whether expressed transiently in COS-1 cells or produced in and purified from Escherichia coli, both mutant enzymes were insensitive to activation by glucose-6-P. The effect of phosphorylation was studied in two ways. Purified, recombinant glycogen synthase was directly phosphorylated by casein kinase 2 and glycogen synthase kinase 3, under conditions that inactivate the wild-type enzyme. In addition, phosphorylation sites were converted to Ala by mutagenesis in wild-type and in the glucose-6-P desensitized mutants expressed in COS-1 cells. Phosphorylation inactivated the R578R579R581A mutant but had little effect on the R585R587R590A. This result was surprising since phosphorylation had the opposite effects on the corresponding yeast enzyme mutants. The results confirm that the region of glycogen synthase, Arg-578-Arg-590, is required for activation by glucose-6-P and suggest that it is part of a sensitive and critical switch involved in transitions between different conformational states. However, the role must differ subtly between the mammalian and the yeast enzymes.     

Transport of the yeast ATP synthase beta-subunit into mitochondria. Effects of amino acid substitutions on targeting.

We have isolated the yeast ATP2 gene encoding the beta-subunit of mitochondrial ATP synthase and determined its nucleotide sequence. A fusion between the N-terminal 15 amino acid residues of beta-subunit and the mouse cytosolic protein dihydrofolate reductase (DHFR) was transcribed and translated in vitro and found to be transported into isolated yeast mitochondria. A fusion with the first 35 amino acid residues of beta-subunit attached to DHFR was not only transported but also proteolytically processed by a mitochondrial protease. Amino acid substitutions were introduced into the N-terminal presequence of the beta-subunit by bisulphite mutagenesis of the corresponding DNA. The effects of these mutations on mitochondrial targeting were assessed by transport experiments in vitro using DHFR fusion proteins. All of the mutants, harbourin from one to six amino acid substitutions in the first 14 residues of the presequence, were transported into mitochondria, though at least one of them (I8) was transported and proteolytically processed at a much reduced rate. The I8 mutant beta-subunit also exhibited poor transport and processing in vivo, and expression of this mutant polypeptide failed to complement the glycerol- phenotype of a yeast ATP2 mutant. More remarkably, the expression of I8 beta-subunit induced a more general growth defect in yeast, possibly due to interference with the transport of other, essential, mitochondrial proteins.     

High-throughput site-directed mutagenesis using oligonucleotides synthesized on DNA chips

Site-directed mutagenesis has greatly helped researchers both to understand the precise role of specific residues in coding sequences and to generate variants of proteins that have acquired new characteristics. Today's demands for more complete functional cartographies of proteins and advances in selection and screening technologies require that site-directed mutagenesis be adapted for high-throughput applications. We describe here the first generation of a library of single and multiple site-directed mutants using a mixture of oligonucleotides synthesized on DNA chips. We have used the human interleukin 15 (IL15) gene as a model, of which 37 codons were simultaneously targeted for substitution by any of eight possible codons. Ninety-six clones were sequenced, exhibiting a broad spectrum of targeted substitutions over the whole gene length with no unwanted mutations. Libraries produced using such pools of oligonucleotides open new perspectives to direct the evolution of proteins in vitro, by enabling the simple, rapid, and cost-effective generation of large tailor-made genetic diversities from any gene.     

Temperature-sensitive mutants of hsp82 of the budding yeast Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae has two HSP90-related genes per haploid genome, HSP82 and HSC82. Random mutations were induced in vitro in the HSP82 gene by treatment of the plasmid with hydroxylamine. Four temperature-sensitive (ts) mutants and one simultaneously is and cold-sensitivie (cs) mutant were then selected in a yeast strain in which HSC82 had previously been disrupted. The mutants were found to have single base changes in the coding region, which caused single amino acid substitutions in the HSP82 protein. All of these mutations occurred in amino acid residues that are well conserved among HSP90-related proteins of various species from Escherichia coli to human. Various properties including cell morphology, macromolecular syntheses and thermosensitivity were examined in each mutant at both the permissive and nonpermissive temperatures. The mutations in HSP82 caused pleiotropic effects on these properties although the phenotypes exhibited at the nonpermissive temperature varied among the mutants.     

Temperature-sensitive mutants of hsp82 of the budding yeast Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae has two HSP90-related genes per haploid genome, HSP82 and HSC82. Random mutations were induced in vitro in the HSP82 gene by treatment of the plasmid with hydroxylamine. Four temperature-sensitive (ts) mutants and one simultaneously is and cold-sensitivie (cs) mutant were then selected in a yeast strain in which HSC82 had previously been disrupted. The mutants were found to have single base changes in the coding region, which caused single amino acid substitutions in the HSP82 protein. All of these mutations occurred in amino acid residues that are well conserved among HSP90-related proteins of various species from Escherichia coli to human. Various properties including cell morphology, macromolecular syntheses and thermosensitivity were examined in each mutant at both the permissive and nonpermissive temperatures. The mutations in HSP82 caused pleiotropic effects on these properties although the phenotypes exhibited at the nonpermissive temperature varied among the mutants.     

Identification of a receptor binding site in the carboxyl terminus of human interleukin-6.

To identify a receptor binding site of human interleukin-6 (IL-6), we created a library of IL-6 variants with single amino acid substitutions in the last 15 residues (171-185) in the COOH terminus of IL-6. Twenty-seven IL-6 variants were tested for biological activity on a human hepatoma and a mouse hybridoma cell line. Most variants were additionally tested in a receptor binding assay using a human myeloma cell line. Several single amino acid substitutions in the COOH terminus of IL-6 were found to decrease biological activity significantly. This is especially seen in variants with amino acid substitutions that alter the postulated amphipathical alpha-helix structure between residues 178 and 183. The two highly conserved Arg residues at positions 180 and 183 seem to play a very important role in biological activity. The loss of biological activity in all inactive variants is completely paralleled by a decrease of IL-6 receptor binding, as determined by competition binding experiments. One mutant (Leu171) displayed a higher activity on human cells and a higher binding affinity to the receptor and can be considered an IL-6 agonist. It is concluded that the amphipathical alpha-helix structure in the COOH terminus of IL-6 is critical for ligand receptor interaction. Furthermore, the region between residues Ser178 and Arg183 (Ser-Leu-Arg-Ala-X-Arg) is identified as a receptor binding site in the COOH terminus of human IL-6.     

A pivotal role of Zn-binding residues in the function of the copper chaperone for SOD1

A Cu chaperone for SOD1 (CCS) is required for the incorporation of copper ion into the protein. To investigate the roles of the conserved metal-binding residues in CCS, we introduced amino acid substitutions into human CCS and examined the function of the mutant CCS by transforming a mutant yeast strain, SY2950, which lacks the lys7 gene, a yeast orthologue of the mammalian CCS. Mutant CCS in which amino acid residues His147 and Asp167 were substituted by Ala exhibited a decreased ability to complement the growth of SY2950 under Lys-deficient conditions. This is because the mutations made the human CCS function in a less efficient manner, especially under metal-restricted conditions, leaving Cu,Zn-SOD in an apo-form. Since the His and Asp residues are both responsible for binding Zn which would serve to maintain the folded structure, the structural integrity supported by the coordinated Zn ion would be essential for CCS function.     

Functional interactions between residues in the S1, S4, and S5 domains of Kv2.1

The voltage-gated potassium channel subunit Kv2.1 forms heterotetrameric channels with the silent subunit Kv6.4. Chimeric Kv2.1 channels containing a single transmembrane segment from Kv6.4 have been shown to be functional. However, a Kv2.1 chimera containing both S1 and S5 from Kv6.4 was not functional. Back mutation of individual residues in this chimera (to the Kv2.1 counterpart) identified four positions that were critical for functionality: A200V and A203T in S1, and T343M and P347S in S5. To test for possible interactions in Kv2.1, we used substitutions with charged residues and tryptophan for the outermost pair 203/347. Combinations of substitutions with opposite charges at both T203 and S347 were tolerated but resulted in channels with altered gating kinetics, as did the combination of negatively charged aspartate substitutions. Double mutant cycle analysis with these mutants indicated that both residues are energetically coupled. In contrast, replacing both residues with a positively charged lysine together (T203K + S347K) was not tolerated and resulted in a folding or trafficking deficiency. The nonfunctionality of the T203K + S347K mutation could be restored by introducing the R300E mutation in the S4 segment of the voltage sensor. These results indicate that these specific S1, S4, and S5 residues are in close proximity and interact with each other in the functional channel, but are also important determinants for Kv2.1 channel maturation. These data support the view of an anchoring interaction between S1 and S5, but indicate that this interaction surface is more extensive than previously proposed.     

DNA sequence analysis of the ade6 gene of Schizosaccharomyces pombe. Wild-type and mutant alleles including the recombination host spot allele ade6-M26

The gene ade6 is located on chromosome III of the fission yeast Schizosaccharomyces pombe. It codes for the enzyme phosphoribosylaminoimidazole carboxylase involved in purine biosynthesis. A DNA fragment of 3043 nucleotides has been sequenced. It complements ade6 mutations when present on plasmids. An uninterrupted open reading frame of 552 amino acid residues was identified. A method for the cloning of chromosomal mutations by repair of gapped replication vectors in vivo has been developed. Twelve ade6 mutant alleles have been isolated. The sequence alterations of four mutant alleles have been determined. Among them are the ade6-M26 recombination hot spot mutation and the nearby ade6-M375 control mutation. Both are G to T base substitutions, converting adjacent glycine codons to TGA termination codons. They are suppressed by defined tRNA nonsense suppressors of the UGA type. The ade6-M26 mutation leads to a tenfold increase of the occurrence of conversion tetrads in comparison with other ade6 mutations. Possible explanations for the M26-induced increase of recombination frequency are discussed in relation to specific features of the nucleotide sequence identified in the region of the M26 mutation.