Article Cite This: Biochemistry XXXX, XXX, XXX−XXX
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Hydrophobic Residue in Escherichia coli Thioredoxin Critical for the Processivity of T7 DNA Polymerase Seung-Joo Lee, Ngoc Q. Tran, Joseph Lee, and Charles C. Richardson* Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, United States
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S Supporting Information *
ABSTRACT: Bacteriophage T7 uses the thioredoxin of its host, Escherichia coli, to enhance the processivity of its DNA polymerase, a requirement for the growth of phage T7. The evolutionarily conserved structure and high degree of homology of amino acid sequence of the thioredoxin family imply that homologues from other organisms might also interact with T7 DNA polymerase to support the phage growth. Despite the structural resemblance, human thioredoxin, whose X-ray crystallographic structure overlaps with that of the E. coli protein, cannot support T7 phage growth. It does not form a complex with T7 DNA polymerase as determined by surface plasmon resonance and thus does not increase the processivity. Homologous scanning analysis using this nonfunctional homologue reveals that the 60 Nterminal and the 12 C-terminal amino acid residues of E. coli thioredoxin can be substituted for its human counterpart without significantly affecting phage growth. Comparison of chimeric thioredoxins, followed by site-directed mutagenesis, identifies leucine 95 as a critical element. This residue may contribute to hydrophobic interactions with the thioredoxin-binding loop of the polymerase; levels of DNA binding and thus nucleotide polymerization are significantly decreased in the absence of this residue. The results suggest that the specific interactions at the interface of thioredoxin and DNA polymerase, rather than the overall structure, are important in the interactions that promote high processivity.
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perform efficient amplification of its genome and thus does not grow on E. coli strains lacking thioredoxin.3 An X-ray crystallographic structure of E. coli thioredoxin−T7 DNA polymerase complexed with a DNA template/primer in a polymerization mode provides insight into the mechanism by which thioredoxin bestows processivity on the polymerase2 (Figure 1B). T7 DNA polymerase has a canonical right-handed shape structure similar to those of other family I polymerases. The structure differs however by the presence of an additional 76-amino acid residue loop in the thumb region. This loop, designated the thioredoxin-binding domain (TBD), makes wide-ranging contacts with E. coli thioredoxin. The fact that a portion of the flexible TBD is disordered in the structure implicates a dynamic interaction between the two proteins. E. coli DNA polymerase I, a homologue of T7 DNA polymerase, lacks the TBD and has a relatively low processivity. However, insertion of the T7 TBD at the position homologous to T7 DNA polymerase converts the nonprocessive E. coli polymerase to a processive enzyme in the presence of thioredoxin, thus showing that TBD−thioredoxin interaction is responsible for the processive polymerization of nucleotides.8 Most processivity factors associated with replicative DNA polymerases encircle the DNA. However, the crystal structure reveals the polymerase−thioredoxin complex poised over the
hioredoxins (trx) are ubiquitous proteins involved in redox reactions during various cellular events. Coupled with thioredoxin reductase, thioredoxin reduces or oxidizes a disulfide bond in target proteins, a reaction mediated by its own disulfide bond in the active site. Consequently, thioredoxins are involved in facilitating protein folding, regulation of oxidation stress, and signaling. They also play a role as a hydrogen donor for reduction of ribonucleotides by ribonucleotide reductase.1 Such important roles of thioredoxin are performed by a stable structure designated the thioredoxin fold that is evolutionarily conserved throughout many species. Alternating α helix/β strand sandwich sequences form a stable central scaffold composed of four β strands flanked by α helices. The active site containing a conserved Cys-Gly-ProCys sequence protrudes to the side for easy access of interacting counterparts (Figure 1A). Escherichia coli thioredoxin is unique in that it plays a critical role in the life cycles of bacteriophage T73 and filamentous phages such as f1.4 Its abundance in the periplasm1 provides reducing conditions for facilitating assembly of filamentous phage.5 Thioredoxin is essential for T7 DNA replication. It forms a stable 1:1 complex with phage T7-encoded DNA polymerase with an apparent binding affinity of 5 nM. This interaction increases the processivity of nucleotide polymerization 100-fold and markedly increases the macroscopic rate of DNA synthesis.6 In addition, the binding of thioredoxin to the polymerase configures motifs in the polymerase that facilitate its interactions with the helicase and ssDNA-binding protein of T7.7 In the absence of thioredoxin, the phage cannot © XXXX American Chemical Society
Received: March 21, 2018 Revised: June 18, 2018
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DOI: 10.1021/acs.biochem.8b00341 Biochemistry XXXX, XXX, XXX−XXX
Article
Biochemistry
Figure 1. Structure and amino acid sequence alignment of thioredoxins. (A) Superimposed structures of thioredoxins from E. coli (gray) and human (red) (root-mean-square deviation of 0.958). Secondary structural motifs are labeled. Side chains of the two cysteines in the active site are colored yellow and magenta. (B) X-ray crystallographic structure of the T7 DNA polymerase and E. coli thioredoxin complex bound to a primer/ template.2 Subdomains of T7 DNA polymerase shown in the space-filing model are designated by different colors, and thioredoxin is highlighted with a ribbon model. The inset is an expanded view of the thioredoxin-bound portion rotated 180°. (C) Alignment of amino acid sequences of thioredoxins investigated in this study. Gray and red colors indicate sequences of thioredoxin from E. coli and human, respectively. Replaced or inserted residues are indicated, and dashes represent deleted residues. Secondary structural elements of thioredoxin are shown atop the aligned sequence.
affecting binding to the polymerase arose from studies of mutations that affected growth of f1 phage; no systematic examination of amino acid changes that affect binding to T7 DNA polymerase is available. The large surface of thioredoxin that interacts with the TBD of the polymerase makes it difficult to precisely locate critical connections. The extended and flexible nature of the TBD adds complexity in decoding molecular details.a Considerable information about the interaction of E. coli thioredoxin with T7 DNA polymerase and its effect on the processivity of the polymerization reaction has been gathered. However, as mentioned above, the role of thioredoxin in T7 DNA replication is not limited to its effect on processivity; the binding of thioredoxin to the TBD configures the TBD such that it can interact with the helicase and ssDNA-binding protein.7 The recruitment of helicase to the replisome has a dramatic effect on the overall processivity of replisome movement. The snapshot of the polymerase−thioredoxin complex bound to DNA has been extremely helpful in studies of the T7 replisome, but in vivo, the structures are dynamic as nucleoside triphosphates are recruited to the active site and the polymerase alters its binding affinity to move to the next site in the template. Consequently, it is extremely difficult to predict from existing structures of thioredoxin precisely which regions and residues are important in its interaction with the polymerase. At some point, T7 DNA polymerase selected the abundant and highly conserved E. coli thioredoxin as an
DNA a considerable distance from the active site of the polymerase.2 It has been proposed that the thioredoxin−TBD complex located in the thumb subdomain swings down over the DNA such that it essentially forms a lid over the DNA to increase its affinity for DNA. In support of this hypothesis, the presence of thioredoxin increases the level of protection of the duplex portion of the primer/template from the nuclease from 9 to 16 bp.9 Early studies of the interaction of thioredoxin with T7 DNA polymerases identified several amino acid changes in thioredoxin that affected binding. For example, substitution of Gly-92 for Asp abolishes the ability of thioredoxin to support T7 phage growth and to stimulate T7 DNA polymerase activity.10 A similar change of Gly-74 to Asp results in a moderately negative effect on both phage growth and DNA polymerase activity. Interestingly, suppressor phages that overcome the defect from G74D thioredoxin were isolated and contained alterations in gene 5 of the phage that encodes DNA polymerase.11 Further biochemical analysis found that the defect of G74D thioredoxin can be compensated by alteration of Glu-319 to Lys in the polymerase, suggesting the two residues are contact points in the polymerase−thioredoxin complex.12 Cys-32 and Cys-35, two cysteines critical for the reduction activity of thioredoxin, can be replaced with either alanine or serine without significantly affecting the role of thioredoxin in interacting with T7 DNA polymerase.10 However, most of the amino acid substitutions in thioredoxin B
DOI: 10.1021/acs.biochem.8b00341 Biochemistry XXXX, XXX, XXX−XXX
Article
Biochemistry
DNA Polymerase Assays. The circular DNA template used for DNA synthesis was prepared by annealing a 24-mer oligonucleotide (M13 primer −47) to M13 mp18 ssDNA at a molar ratio of 1:1. The DNA polymerase reaction mixture for the primed M13 ssDNA contained 40 mM Tris-HCl (pH 7.5), 50 mM NaCl, 10 mM MgCl2, 5 mM DTT, dGTP, dATP, dCTP, and [3H]dTTP (250 μM each, 10 cpm/pmol), 20 nM primed M13 ssDNA, and 5 nM T7 DNA polymerase with a 100-fold excess of thioredoxin. The reactions were performed at 37 °C and stopped by addition of EDTA to a final concentration of 25 mM. Aliquots of reaction mixtures were spotted on DE81 ion exchange filters. Unincorporated radiolabeled nucleotides were removed by washing the filters with three successive 5 min washes in 300 mM ammonium formate (pH 8.0). Filters were dried, and the amount of [3H]dTMP incorporated into DNA was determined by liquid scintillation counting. The homopolymer DNA substrate (dA200/dT17) was prepared by annealing a 17-nucleotide oligo-dT to a 200nucleotide poly-dA. Reactions for monitoring DNA polymerase activity on the homopolymer substrate were similar to those for primed M13 ssDNA except that dA200/dT17 was added at a concentration of 200 nM, resulting in a 40-fold molar excess of DNA over polymerase, and the reaction mixtures were incubated at 25 °C. Examination of Reaction Products on an Alkaline Agarose Gel. To determine the processivity of polymerase reactions with various thioredoxins, reaction products from DNA polymerase assays were separated on an alkaline agarose gel. The primer was radioactively labeled at the 5′-end by incubating with [γ-32P]ATP and T4 polynucleotide kinase (New England Biolabs) at 37 °C for 30 min followed by heating at 65 °C for 10 min. The radiolabeled primer was annealed to circular M13 ssDNA in 50 mM Tris-HCl (pH 7.5) and 50 mM NaCl, and the resulting primed M13 DNA was further purified using S-300 Microspin columns (GE Healthcare). The DNA polymerase reaction mixture (100 μL) contained 50 mM Tris-HCl (pH 7.5), 5 mM DTT, 50 mM NaCl, 10 mM MgCl2, four dNTPs (250 μM each), 5 nM T7 DNA polymerase, a 100-fold excess of thioredoxin, and 20 nM M13 ssDNA annealed to the 32P-labeled primer. After reaction mixtures were incubated at 37 °C for the indicated times, aliquots (20 μL) were removed and the reaction was terminated by adding EDTA to a final concentration of 25 mM. The reaction products were separated by electrophoresis on a 0.6% alkaline agarose gel. The gel was dried and exposed to a phosphoimager plate followed by scanning with a Fuji BAS 1000 Bio-Imaging Analyzer. Determination of Protein Binding by Surface Plasmon Resonance. Physical interaction between thioredoxin and T7 DNA polymerase was measured by surface plasmon resonance using a Biacore 3000 instrument in two different setups. When thioredoxin was immobilized, the indicated amounts of thioredoxin were immobilized on a Nicharged NTA chip via a histidine tag at the N-terminus in binding buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 10 mM MgCl2. Sixty microliters of 430 nM T7 DNA polymerase was passed over the chip at a flow rate of 40 μL/min. The binding signal was adjusted by subtracting the background from a negative control flow cell containing no thioredoxin. The chip was regenerated by washing with a buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl,
accessory protein. Inspection of the E. coli and other conserved thioredoxin sequences does not provide sufficient information about their relative affinities for T7 DNA polymerase. Consequently, we consider it important and informative to examine the ability of other thioredoxins to support T7 DNA replication and to interact with the polymerase. Such information provides insight into interactions not predicted by the structure and provides insight into the evolution of the wide family of thioredoxins. In our study, we examined interaction of T7 DNA polymerase with E. coli thioredoxin and human thioredoxin, whose structures resemble each other. Chimeric thioredoxins in which a portion of E. coli thioredoxin was replaced with the homologous part of its human counterpart were also constructed and examined.
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MATERIALS AND METHODS Construction of Plasmids. Thioredoxin genes from either E. coli (trxA) or human (TXN) were subcloned into pET19b between NdeI and EcoRI sites using pTrxA or pGEX-htrx (a generous gift from P. Patwari, Brigham and Women’s Hospital, Boston, MA), respectively, as templates for polymerase chain reaction (PCR). Alterations in the thioredoxin genes were introduced by the megaprimer PCR method13 using a primer containing the designated mutation. The presence of the intended alteration was confirmed by DNA sequence analysis. Overproduction and Purification of Proteins. T7 gene 5 DNA polymerase and wild-type E. coli thioredoxin were prepared as described previously.14 Most thioredoxins used in this study contained 10 histidine residues at their N-termini and were overproduced from E. coli strain A307 (DE3) lacking trxA. E. coli cells containing the thioredoxin-expressing plasmid were cultured until the A600 reached ∼1. After the addition of isopropyl β-D-1-thiogalactopyranoside to a final concentration of 1 mM, the culture was incubated for a further 3 h at 37 °C. The cells were lysed by three cycles of freezing and thawing in the presence of lysozyme, and the clear lysate obtained by centrifugation was incubated with Ni-NTA resin for 1 h in the cold room. The resin was serially washed with buffer A [20 mM Tris-HCl (pH 7.5) and 150 mM NaCl] containing 0, 25, and 100 mM imidazole and finally eluted with buffer A containing 500 mM imidazole. The eluted protein was precipitated by addition of 5 g of ammonium sulfate per 10 mL of eluent. Precipitated proteins collected by centrifugation were further purified on a G-50 gel filtration column in buffer B [50 mM Tris-HCl (pH 7.5) and 1 mM EDTA]. After fractions containing thioredoxin had been collected, the protein was precipitated with ammonium sulfate as described above, redissolved in buffer B, and dialyzed against buffer B containing 50% glycerol. T7 Phage Growth Complementation Assay. Plasmids expressing various thioredoxins were transformed into E. coli strain A307 lacking trxA. Cells containing the thioredoxinexpressing plasmid were plated on LB plates, and aliquots of serially diluted T7 phage were spotted to determine a proper range of phage concentrations. In the plaque assay, 0.3 mL of cells in the log phase and 0.1 mL of properly diluted phage were mixed with 3 mL of soft agar and plated on a LB plate. The plaques that appeared from overnight incubation of the plates at 37 °C were counted and normalized against those obtained with the wild-type E. coli thioredoxin-expressing plasmid. C
DOI: 10.1021/acs.biochem.8b00341 Biochemistry XXXX, XXX, XXX−XXX
Article
Biochemistry
throughout the thioredoxin family raise the possibility that a homologue from other species could replace E. coli thioredoxin in its interaction with T7 DNA polymerase. The amino acid sequences of human thioredoxin and E. coli thioredoxin are >50% homologous [33 identical and 42 similar residues from ClustalW analysis (Supporting Information)]. Superimposition of the X-ray crystallographic structures of the two proteins shows their structural similarities (Figure 1A). The potential replacement of E. coli thioredoxin with the human homologue was first examined by its ability to support T7 phage growth in a complementation assay. In this assay, the thioredoxin gene to be tested is located on a plasmid under control of a T7 promoter harbored in E. coli lacking thioredoxin. Because interaction of thioredoxin with T7 DNA polymerase is essential for phage growth, only a functional thioredoxin produced from the plasmid upon T7 phage infection allows plaque formation. As shown in Table 1, E. coli thioredoxin supports phage growth regardless of the presence of a histidine tag whereas human thioredoxin does not (Table 1).
and 350 mM EDTA. When T7 DNA polymerase was immobilized, approximately 5000 RU of the protein was immobilized on the CM-5 chip and 10 μM thioredoxin was passed over the chip at a flow rate of 10 μL/min in binding buffer containing 20 mM potassium phosphate (pH 7.4), 10 mM NaCl, and 5 mM DTT. The binding signal was adjusted by subtracting the background from a negative control flow cell containing no T7 DNA polymerase. The chip was regenerated by washing twice with 30 μL of 1 M NaCl. The binding signal was analyzed with Biacore T200 evaluation software 3.0. Determination of DNA Binding by Surface Plasmon Resonance. Binding of T7 DNA polymerase to a template/ primer in the presence of various thioredoxins was measured by surface plasmon resonance using a Biacore 3000 instrument similar to that described previously.7 A 26-mer template (5′CAGTG ACGGG TCGTT TATCG TCGGC A-3′) was annealed to a 19-mer primer (5′-TGCCG ACGAT AAACG ACCC-3′) and immobilized on a SA chip via a biotin tag at the 3′-end of the template (150 RU) in binding buffer containing 20 mM HEPES (pH 7.5), 200 mM potassium glutamate, 10 mM MgCl2, 5 mM DTT, and 1% glycerol. T7 DNA polymerase (50 nM) was passed over the chip at a flow rate of 10 μL/min in the absence or presence of 2 μM thioredoxin. The binding signal was adjusted by subtracting the background from a negative control flow cell containing no DNA. The chip was regenerated by washing with 100 μL of 1 M NaCl. The binding signal was analyzed by Biacore T200 evaluation software 3.0. Circular Dichroism (CD) Spectroscopy. CD measurements were taken using a Jasco J-810 spectropolarimeter with a 1 mm path-length quartz cuvette at 25 °C. A series of five scans were acquired between wavelengths of 200 and 260 nm for each thioredoxin at concentrations of 1.25, 2.5, and 5 μM. Each series was then averaged and normalized against a buffer baseline. To minimize any potential absorption from the solvent, each thioredoxin was dialyzed against a buffer consisting of 10 mM potassium phosphate (pH 7.5) and 1 mM DTT before data collection. Determination of the Reduction Activity of Thioredoxin. Reduction of insulin by thioredoxin leads to cleavage of the two interchain disulfide bridges of insulin,15 resulting in a white precipitate due to insolubility.16 This phenomenon was used to examine the reduction activity of the thioredoxins by measuring the rate of precipitation of reduced insulin at 650 nm. An insulin solution (1 mg/mL) was freshly prepared in 50 mM Tris-HCl (pH 7.5) and 2 mM EDTA. An assay mixture of 100 μL of insulin and thioredoxin (3 μM) supplemented with water to give a final volume of 150 μL was placed in a cuvette and served as a blank control. The reaction was initiated by adding 1 mM DTT to the cuvette, thoroughly mixing the contents, and placing the cuvette in a NanoPhotometer P-360 (Implen). The kinetics of the change in optical density at 650 nm (A650) was automatically recorded with a 1 min interval for 45 min. The non-enzymatic reduction of insulin by DTT was also recorded as a negative control reaction without thioredoxin. The reduction activity of thioredoxin was compared by determining the increased rate of precipitation at 650 nm per minute (ΔA650 min−1) within the linear range.
Table 1. Ability of Thioredoxin To Complement the Growth of T7 Phagea thioredoxin none E. coli (native)c E. coli human chimeric HN1 HN2 HN3 HN4 HN5 HC1 HC2 HC3 HC4 HN1+C3 deletion/insertion E. coli ΔL95 E. coli ΔS96 E. coli L95N/ΔS96 human insL95
efficiency of platingb
plaque sizeb
−8
