TnTIN and TnTAP: Mini-transposons for site-specific proteolysis in vivo

Tobacco etch virus (TEV) protease recognizes a 7-aa consensus sequence, Glu-Xaa-Xaa-Tyr-Xaa-Gln-Ser, where Xaa can be almost any amino acyl residue. Cleavage occurs between the conserved Gln and Ser residues. Because of its distinct specificity, TEV protease can be expressed in the cytoplasm without interfering with viability. Polypeptides that are not natural substrates of TEV protease are proteolyzed if they carry the appropriate cleavage site. Thus, this protease can be used to study target proteins in their natural environment in vivo, as well as in vitro. We describe two Tn5-based mini-transposons that insert TEV protease cleavage sites at random into target proteins. TnTIN introduces TEV cleavage sites into cytoplasmic proteins. TnTAP facilitates the same operation for proteins localized to the bacterial cell envelope. By using two different target proteins, SecA and TolC, we show that such modified proteins can be cleaved in vivo and in vitro by TEV protease. Possible applications of the site-specific proteolysis approach are topological studies of soluble as well as of inner and outer membrane proteins, protein inactivation, insertion mutagenesis experiments, and protein tagging.

Expression of myogenin during embryogenesis is controlled by Six/sine oculis homeoproteins through a conserved MEF3 binding site

Myogenin, one of the MyoD family of proteins, is expressed early during somitogenesis and is required for myoblast fusion in vivo. Previous studies in transgenic mice have shown that a 184-bp myogenin promoter fragment is sufficient to correctly drive expression of a β-galactosidase transgene during embryogenesis. We show here that mutation of one of the DNA motifs present in this region, the MEF3 motif, abolished correct expression of this β-galactosidase transgene. We have found that the proteins that bind to the MEF3 site are homeoproteins of the Six/sine oculis family. Antibodies directed specifically against Six1 or Six4 proteins reveal that each of these proteins is present in the embryo when myogenin is activated and constitutes a muscle-specific MEF3-binding activity in adult muscle nuclear extracts. Both of these proteins accumulate in the nucleus of C2C12 myogenic cells, and transient transfection experiments confirm that Six1 and Six4 are able to transactivate a reporter gene containing MEF3 sites. Altogether these results establish Six homeoproteins as a family of transcription factors controlling muscle formation through activation of one of its key regulators, myogenin.

DNA polymerase active site is highly mutable: Evolutionary consequences

DNA polymerases contain active sites that are structurally superimposable and highly conserved in sequence. To assess the significance of this preservation and to determine the mutational burden that active sites can tolerate, we randomly mutated a stretch of 13 amino acids within the polymerase catalytic site (motif A) of Thermus aquaticus DNA polymerase I. After selection, by using genetic complementation, we obtained a library of approximately 8,000 active mutant DNA polymerases, of which 350 were sequenced and analyzed. This is the largest collection of physiologically active polymerase mutants. We find that all residues of motif A, except one (Asp-610), are mutable while preserving wild-type activity. A wide variety of amino acid substitutions were obtained at sites that are evolutionarily maintained, and conservative substitutions predominate at regions that stabilize tertiary structures. Several mutants exhibit unique properties, including DNA polymerase activity higher than the wild-type enzyme or the ability to incorporate ribonucleotide analogs. Bacteria dependent on these mutated polymerases for survival are fit to replicate repetitively. The high mutability of the polymerase active site in vivo and the ability to evolve altered enzymes may be required for survival in environments that demand increased mutagenesis. The inherent substitutability of the polymerase active site must be addressed relative to the constancy of nucleotide sequence found in nature.

Identification of eIF2Bγ and eIF2γ as cofactors of hepatitis C virus internal ribosome entry site-mediated translation using a functional genomics approach

The 5′-untranslated region of hepatitis C virus (HCV) is highly conserved, folds into a complex secondary structure, and functions as an internal ribosome entry site (IRES) to initiate translation of HCV proteins. We have developed a selection system based on a randomized hairpin ribozyme gene library to identify cellular factors involved in HCV IRES function. A retroviral vector ribozyme library with randomized target recognition sequences was introduced into HeLa cells, stably expressing a bicistronic construct encoding the hygromycin B phosphotransferase gene and the herpes simplex virus thymidine kinase gene (HSV-tk). Translation of the HSV-tk gene was mediated by the HCV IRES. Cells expressing ribozymes that inhibit HCV IRES-mediated translation of HSV-tk were selected via their resistance to both ganciclovir and hygromycin B. Two ribozymes reproducibly conferred the ganciclovir-resistant phenotype and were shown to inhibit IRES-mediated translation of HCV core protein but did not inhibit cap-dependent protein translation or cell growth. The functional targets of these ribozymes were identified as the gamma subunits of human eukaryotic initiation factors 2B (eIF2Bγ) and 2 (eIF2γ), respectively. The involvement of eIF2Bγ and eIF2γ in HCV IRES-mediated translation was further validated by ribozymes directed against additional sites within the mRNAs of these genes. In addition to leading to the identification of cellular IRES cofactors, ribozymes obtained from this cellular selection system could be directly used to specifically inhibit HCV viral translation, thereby facilitating the development of new antiviral strategies for HCV infection.

Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity

Single-chain antibody mutants have been evolved in vitro with antigen-binding equilibrium dissociation constant Kd = 48 fM and slower dissociation kinetics (half-time > 5 days) than those for the streptavidin–biotin complex. These mutants possess the highest monovalent ligand-binding affinity yet reported for an engineered protein by over two orders of magnitude. Optimal kinetic screening of randomly mutagenized libraries of 105–107 yeast surface-displayed antibodies enabled a >1,000-fold decrease in the rate of dissociation after four cycles of affinity mutagenesis and screening. The consensus mutations are generally nonconservative by comparison with naturally occurring mouse Fv sequences and with residues that do not contact the fluorescein antigen in the wild-type complex. The existence of these mutants demonstrates that the antibody Fv architecture is not intrinsically responsible for an antigen-binding affinity ceiling during in vivo affinity maturation.

Directed evolution of a (βα)8-barrel enzyme to catalyze related reactions in two different metabolic pathways

Enzymes participating in different metabolic pathways often have similar catalytic mechanisms and structures, suggesting their evolution from a common ancestral precursor enzyme. We sought to create a precursor-like enzyme for N′-[(5′-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (ProFAR) isomerase (HisA; EC 5.3.1.16) and phosphoribosylanthranilate (PRA) isomerase (TrpF; EC 5.3.1.24), which catalyze similar reactions in the biosynthesis of the amino acids histidine and tryptophan and have a similar (βα)8-barrel structure. Using random mutagenesis and selection, we generated several HisA variants that catalyze the TrpF reaction both in vivo and in vitro, and one of these variants retained significant HisA activity. A more detailed analysis revealed that a single amino acid exchange could establish TrpF activity on the HisA scaffold. These findings suggest that HisA and TrpF may have evolved from an ancestral enzyme of broader substrate specificity and underscore that (βα)8-barrel enzymes are very suitable for the design of new catalytic activities.

A method for directed evolution and functional cloning of enzymes

A general scheme is described for the in vitro evolution of protein catalysts in a biologically amplifiable system. Substrate is covalently and site specifically attached by a flexible tether to the pIII coat protein of a filamentous phage that also displays the catalyst. Intramolecular conversion of substrate to product provides a basis for selecting active catalysts from a library of mutants, either by release from or attachment to a solid support. This methodology has been developed with the enzyme staphylococcal nuclease as a model. An analysis of factors influencing the selection efficiency is presented, and it is shown that phage displaying staphylococcal nuclease can be enriched 100-fold in a single step from a library-like ensemble of phage displaying noncatalytic proteins. Additionally, this approach should allow one to functionally clone natural enzymes, based on their ability to catalyze specific reactions (e.g., glycosyl transfer, sequence-specific proteolysis or phosphorylation, polymerization, etc.) rather than their sequence- or structural homology to known enzymes.

Mutagenesis of the regulatory domain of phenylalanine hydroxylase

The regulatory domain of phenylalanine hydroxylase (PAH, EC 1.14.16.1) consists of more than 100 amino acids at the N terminus, the removal of which significantly activates the enzyme. To study the regulatory properties controlled by the N terminus, a series of truncations and site-specific mutations were made in this region of rat PAH. These enzymes were expressed highly in Escherichia coli and purified through a pterin-conjugated Sepharose affinity column. The removal of the first 26 amino acids of the N terminus increased the activity by about 20-fold, but removal of the first 15 amino acids increased the activity by only 2-fold. Replacing serine-29 of rat PAH with cysteine from the same site of human PAH increased the activity by more than 4-fold. Mutation of serine to other amino acids with varying side chains: alanine, methionine, leucine, aspartic acid, asparagine, and arginine also resulted in significant activation, indicating a serine-specific inhibitory effect. But these site-specific mutants showed 30–40% lower activity when assayed with 6-methyl-5,6,7,8-tetrahydropterin. Stimulation of hydroxylase activity by preincubation of the enzyme with phenylalanine was inversely proportional to the activation state of all these mutants. Combined with recent crystal structures of PAH [Kobe, B. et al. (1999) Nat. Struct. Biol. 6, 442–448; and Erlandsen, H., Bjorgo, E., Flatmark, T. & Stevens, R. C. (2000) Biochemistry 39, 2208–2217], these data suggest that residues 16–26 have a controlling regulatory effect on the activity by interaction with the dihydroxypropyl side chain of (6R)-5,6,7,8-tetrahydrobiopterin. The serine/cysteine switch explains the difference in regulatory properties between human and rat PAH. The N terminus as a whole is important for maintaining rat PAH in an optimum catalytic conformation.

Orthogonal combinatorial mutagenesis: a codon-level combinatorial mutagenesis method useful for low multiplicity and amino acid-scanning protocols

We describe here a method to generate combinatorial libraries of oligonucleotides mutated at the codon-level, with control of the mutagenesis rate so as to create predictable binomial distributions of mutants. The method allows enrichment of the libraries with single, double or larger multiplicity of amino acid replacements by appropriate choice of the mutagenesis rate, depending on the concentration of synthetic precursors. The method makes use of two sets of deoxynucleoside-phosphoramidites bearing orthogonal protecting groups [4,4′-dimethoxytrityl (DMT) and 9-fluorenylmethoxycarbonyl (Fmoc)] in the 5′ hydroxyl. These phosphoramidites are divergently combined during automated synthesis in such a way that wild-type codons are assembled with commercial DMT-deoxynucleoside-methyl-phosphoramidites while mutant codons are assembled with Fmoc-deoxynucleoside-methyl-phosphoramidites in an NNG/C fashion in a single synthesis column. This method is easily automated and suitable for low mutagenesis rates and large windows, such as those required for directed evolution and alanine scanning. Through the assembly of three oligonucleotide libraries at different mutagenesis rates, followed by cloning at the polylinker region of plasmid pUC18 and sequencing of 129 clones, we concluded that the method performs essentially as intended.

Site-specifically located 8-amino-2′-deoxyguanosine: thermodynamic stability and mutagenic properties in Escherichia coli

2-Nitropropane (2-NP), an important industrial solvent and a component of cigarette smoke, is mutagenic in bacteria and carcinogenic in rats. 8-Amino-2′-deoxyguanosine (8-amino-dG) is one of the types of DNA damage found in liver, the target organ in 2-NP-treated rats. To investigate the thermodynamic properties of 8-amino-dG opposite each of the four DNA bases, we have synthesized an 11mer, d(CCATCG*CTACC), in which G* represents the modified base. By annealing a complementary DNA strand to this modified 11mer, four sets of duplexes were generated each containing one of the four DNA bases opposite the lesion. Circular dichroism studies indicated that 8-amino-dG did not alter the global helical properties of natural right-handed B-DNA. The thermal stability of each duplex was examined by UV melting measurements and compared with its unmodified counterpart. For the unmodified 11mer, the relative stability of the complementary DNA bases opposite G was in the order C > T > G > A, as determined from their –ΔG° values. The free energy change of each modified duplex was lower than its unmodified counterpart, except for the G*:G pair that exhibited a higher melting transition and a larger –ΔG° than the G:G duplex. Nevertheless, the stability of the modified 11mer duplex also followed the order C > T > G > A when placed opposite 8-amino-dG. To explore if 8-amino-dG opposite another 8-amino-dG has any advantage in base pairing, a G*:G* duplex was evaluated, which showed that the stability of this duplex was similar to the G*:G duplex. Mutagenesis of 8-amino-dG in this sequence context was studied in Escherichia coli, which showed that the lesion is weakly mutagenic (mutation frequency ∼10–3) but still can induce a variety of targeted and semi-targeted mutations.

Fission Yeast Aip3p (spAip3p) Is Required for an Alternative Actin-directed Polarity Program

Aip3p is an actin-interacting protein that regulates cell polarity in budding yeast. The Schizosaccharomyces pombe-sequencing project recently led to the identification of a homologue of Aip3p that we have named spAip3p. Our results confirm that spAip3p is a true functional homologue of Aip3p. When expressed in budding yeast, spAip3p localizes similarly to Aip3p during the cell cycle and complements the cell polarity defects of an aip3Δ strain. Two-hybrid analysis shows that spAip3p interacts with actin similarly to Aip3p. In fission yeast, spAip3p localizes to both cell ends during interphase and later organizes into two rings at the site of cytokinesis. spAip3p localization to cell ends is dependent on microtubule cytoskeleton, its localization to the cell middle is dependent on actin cytoskeleton, and both patterns of localization require an operative secretory pathway. Overexpression of spAip3p disrupts the actin cytoskeleton and cell polarity, leading to morphologically aberrant cells. Fission yeast, which normally rely on the microtubule cytoskeleton to establish their polarity axis, can use the actin cytoskeleton in the absence of microtubule function to establish a new polarity axis, leading to the formation of branched cells. spAip3p localizes to, and is required for, branch formation, confirming its role in actin-directed polarized cell growth in both Schizosaccharomyces pombe and Saccharomyces cerevisiae.

Two recombination-dependent DNA replication pathways of bacteriophage T4, and their roles in mutagenesis and horizontal gene transfer

Two major pathways of recombination-dependent DNA replication, “join-copy” and “join-cut-copy,” can be distinguished in phage T4: join-copy requires only early and middle genes, but two late proteins, endonuclease VII and terminase, are uniquely important in the join-cut-copy pathway. In wild-type T4, timing of these pathways is integrated with the developmental program and related to transcription and packaging of DNA. In primase mutants, which are defective in origin-dependent lagging-strand DNA synthesis, the late pathway can bypass the lack of primers for lagging-strand DNA synthesis. The exquisitely regulated synthesis of endo VII, and of two proteins from its gene, explains the delay of recombination-dependent DNA replication in primase (as well as topoisomerase) mutants, and the temperature-dependence of the delay. Other proteins (e.g., the single-stranded DNA binding protein and the products of genes 46 and 47) are important in all recombination pathways, but they interact differently with other proteins in different pathways. These homologous recombination pathways contribute to evolution because they facilitate acquisition of any foreign DNA with limited sequence homology during horizontal gene transfer, without requiring transposition or site-specific recombination functions. Partial heteroduplex repair can generate what appears to be multiple mutations from a single recombinational intermediate. The resulting sequence divergence generates barriers to formation of viable recombinants. The multiple sequence changes can also lead to erroneous estimates in phylogenetic analyses.

Site-specific integration by adeno-associated virus.

Adeno-associated virus (AAV) has attracted considerable interest as a potential vector for gene delivery. Wild-type virus is notable for the lack of association with any human disease and the ability to stably integrate its genome in a site-specific manner in a locus on human chromosome 19 (AAVS1). Use of a functional model system for AAV DNA integration into AAVS1 has allowed us to conclude that the recombination event is directed by cellular DNA sequences. Recombinant junctions isolated from our integration assay were analyzed and showed characteristics similar to those found in latently infected cell lines. The minimal DNA signals within AAVS1 required for targeted integration were identified and shown to contain functional motifs of the viral origin of replication. A replication mediated model of AAV DNA integration is proposed.

Identification of three physically and functionally distinct binding sites for C3b in human complement factor H by deletion mutagenesis.

Human complement factor H controls spontaneous activation of complement in plasma and appears to play a role in distinguishing host cells from activators of the alternative pathway of complement. In both mice and humans, the protein is composed of 20 homologous short consensus repeat (SCR) domains. The size of the protein suggests that portions of the structure outside the known C3b binding site (SCR 1-4) possess a significant biological role. We have expressed the full-length cDNA of factor H in the baculovirus system and have shown the recombinant protein to be fully active. Mutants of this full-length protein have now been prepared, purified, and examined for cofactor activity and binding to C3b and heparin. The results demonstrate (i) that factor H has at least three sites that bind C3b, (ii) that one of these sites is located in SCR domains 1-4, as has been shown by others, (iii) that a second site exists in the domain 6-10 region, (iv) that a third site resides in the SCR 16-20 region, and (v) that two heparin binding sites exist in factor H, one near SCR 13 and another in the SCR 6-10 region. Functional assays demonstrated that only the first C3b site located in SCR 1-4 expresses factor I cofactor activity. Mutant proteins lacking any one of the three C3b binding sites exhibited 6- to 8-fold reductions in affinity for C3b on sheep erythrocytes, indicating that all three sites contribute to the control of complement activation on erythrocytes. The identification of multiple functionally distinct sites on factor H clarifies many of the heretofore unexplainable behaviors of this protein, including the heterogeneous binding of factor H to surface-bound C3b, the effects of trypsin cleavage, and the differential control of complement activation on activators and nonactivators of the alternative pathway of complement.

Random mutagenesis of Thermus aquaticus DNA polymerase I: concordance of immutable sites in vivo with the crystal structure.

Expression of Thermus aquaticus (Taq) DNA polymerase I (pol I) in Escherichia, coli complements the growth defect caused by a temperature-sensitive mutation in the host pol I. We replaced the nucleotide sequence encoding amino acids 659-671 of the O-helix of Taq DNA pol I, corresponding to the substrate binding site, with an oligonucleotide containing random nucleotides. Functional Taq pol I mutants were selected based on colony formation at the nonpermissive temperature. By using a library with 9% random substitutions at each of 39 positions, we identified 61 active Taq pol I mutants, each of which contained from one to four amino acid substitutions. Some amino acids, such as alanine-661 and threonine-664, were tolerant of several or even many diverse replacements. In contrast, no replacements or only conservative replacements were identified at arginine-659, lysine-663, and tyrosine-671. By using a library with totally random nucleotides at five different codons (arginine-659, arginine-660, lysine-663, phenylalanine-667, and glycine-668), we confirmed that arginine-659 and lysine-663 were immutable, and observed that only tyrosine substituted for phenylalanine-667. The two immutable residues and the two residues that tolerate only highly conservative replacements lie on the side of O-helix facing the incoming deoxynucleoside triphosphate, as determined by x-ray analysis. Thus, we offer a new approach to assess concordance of the active conformation of an enzyme, as interpreted from the crystal structure, with the active conformation inferred from in vivo function.