Structure-specific DNA-induced Conformational Changes in Taq ...

THE JOURNAL

OF

BIOLOGICAL CHEMISTRY

Vol. 279, No. 37, Issue of September 10, pp. 39146 –39154, 2004 Printed in U.S.A.

Structure-specific DNA-induced Conformational Changes in Taq Polymerase Revealed by Small Angle Neutron Scattering* Received for publication, April 26, 2004, and in revised form, June 30, 2004 Published, JBC Papers in Press, July 7, 2004, DOI 10.1074/jbc.M404565200

Derek L. Ho‡, W. Malcolm Byrnes§, Wu-po Ma¶, Yuan Shi储, David J. E. Callaway储**, and Zimei Bu‡‡§§ From the ‡National Institute of Standards and Technology, Gaithersburg, Maryland 20898, the §Department of Biochemistry and Molecular Biology, Howard University College of Medicine, Washington, D. C. 20059, ¶Third Wave Technologies, Inc., Madison, Wisconsin 53719, 储North Shore/LIJ Research Institute, New York University School of Medicine, Manhasset, New York 11030, and the ‡‡Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111

The DNA polymerase I from Thermus aquaticus (Taq polymerase) performs lagging-strand DNA synthesis and DNA repair. Taq polymerase contains a polymerase domain for synthesizing a new DNA strand and a 5ⴕnuclease domain for cleaving RNA primers or damaged DNA strands. The extended crystal structure of Taq polymerase poses a puzzle on how this enzyme coordinates its polymerase and the nuclease activities to generate only a nick. Using contrast variation solution small angle neutron scattering, we have examined the conformational changes that occur in Taq polymerase upon binding “overlap flap” DNA, a structure-specific DNA substrate that mimics the substrate in strand replacement reactions. In solution, apoTaq polymerase has an overall expanded equilibrium conformation similar to that in the crystal structure. Upon binding to the DNA substrate, both the polymerase and the nuclease domains adopt more compact overall conformations, but these changes are not enough to bring the two active sites close enough to generate a nick. Reconstruction of the three-dimensional molecular envelope from small angle neutron scattering data shows that in the DNAbound form, the nuclease domain is lifted up relative to its position in the non-DNA-bound form so as to be in closer contact with the thumb and palm subdomains of the polymerase domain. The results suggest that a form of structure sensing is responsible for the coordination of the polymerase and nuclease activities in nick generation. However, interactions between the polymerase and the nuclease domains can assist in the transfer of the DNA substrate from one active site to the other.

In addition to its well known in vitro applications in PCR, in vivo the DNA polymerase I from Thermus aquaticus (Taq polymerase) performs nucleotide replacement reactions in DNA repair and RNA primer removal in Okazaki fragment processing during lagging-strand synthesis (1). Taq polymerase also

* This work was supported by an Alzheimer’s Association grant and an IBM Sponsored University Research Grant (to D. J. E. C.) and by National Institutes of Health Grant 2 G12 RR003048 from the National Center for Research Resources (to W. M. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ** To whom correspondence may be addressed: North Shore/LIJ Research Institute, New York University School of Medicine, 350 Community Dr., Manhasset, NY 11030. E-mail: [email protected]. §§ To whom correspondence may be addressed: Fox Chase Cancer Center, Reimann 414, 333 Cottman Ave, Philadelphia, PA 19111. Tel.: 215-728-7051; Fax: 215-728-3574; E-mail: [email protected].

utilizes both its polymerase and 5⬘-nuclease activities in a nucleotide replacement reaction: it catalyzes the upstream addition of deoxynucleoside 5⬘-triphosphates to the 3⬘-hydroxyl terminus of an RNA primer and also cleaves the downstream, single-stranded 5⬘-nucleotide displaced by the growing upstream strand. On a DNA duplex substrate, upstream primer extension and downstream strand excision must be highly coordinated (2) to leave a ligatable nick, instead of a gap or an overhang, on a DNA duplex that is to be sealed later by a DNA ligase. The structure and functional properties of Taq polymerase are similar to those of the polymerase I of Escherichia coli (3). Taq polymerase has two distinct domains: an N-terminal domain (residues 1–289) that has single-stranded 5⬘-nucleotide cleavage activity (5⬘-nuclease domain), and a C-terminal domain (residues 306 – 832) that has DNA polymerase activity (Klentaq domain). Integrated into the Klentaq domain is another domain (residues 306 – 423) that is structurally analogous to the proof editing 3⬘ 3 5⬘-exonuclease domain of E. coli polymerase I, but this domain has no known activity in Taq polymerase. The Klentaq and the 5⬘-nuclease domains are connected by a linker region (residues 290 –305). Structural and functional analysis has revealed that the 5⬘-nuclease of Taq polymerase is a member of a group of structure-specific DNA cleavage nucleases in various viruses, bacteria, archaea, and higher eukaryotes. These 5⬘-nucleases are either physically linked to polymerases as in Taq polymerase and E. coli polymerase I or exist as subunits within complexes that contain discrete polymerase and nuclease activities. Examples of this latter group include the phage-encoded T5 5⬘nucleases (4, 5), archaeal (6) and mammalian flap endonuclease I (7), all of which share an ␣/␤ topology with a common central ␤-sheet and similar surrounding ␣-helices. The active site comprises a cleft formed within the central ␤-sheet; at the bottom of cleft is a set of conserved acidic residues that are essential for binding the three divalent metal ions (two Mn2⫹ ions and one Zn2⫹ ion) required for nuclease activity. The structure of native Taq polymerase (8) shows that there are three metal ions at the active site of the nuclease domain, two Mn2⫹ and a Zn2⫹, consistent with the two-metal ion mechanism proposed for nuclease activity (9). Biochemical studies have shown that the 5⬘-nuclease of Taq polymerase recognizes and endonucleolytically cleaves a structure-specific DNA substrate that has a bifurcated downstream duplex and an upstream template-primer duplex that overlaps the downstream duplex by 1 bp (overlap flap DNA substrate; see Fig. 1B) (10, 11). The 5⬘-nuclease cleaves the unpaired 5⬘-arm of the overlap flap DNA substrate by cutting endonucleolytically at the 1st bp of the duplex at the junction between

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Conformational States in Taq Polymerase the upstream and downstream duplexes (see arrow in Fig. 1B). The T5 5⬘-nuclease and flap endonuclease I also recognize and cleave overlap flap DNA in the same structure-specific manner (12–15), although in the presence of EDTA and in the absence of Mg2⫹, there is no cleavage. In Pyrococcus furiosus flap endonuclease I (16) and T5 5⬘-nuclease (4), a helix-3-turn-helix motif forms a helical clamp that has been proposed to accommodate a threaded unpaired 5⬘-single-stranded nucleic acid. In the original Taq polymerase crystal structure, this arch region is disordered and missing. The authors nevertheless proposed the existence of a loop that forms a hole (8) through which the DNA flap produced by strand displacement can be threaded. Altogether, the evidence so far suggests that recognition and cleavage of the structure-specific DNA substrate is an exclusive characteristic of the 5⬘-nuclease domain, although experiments do also suggest that the polymerase domain can influence the nuclease domain (11, 17, 18). The extended nature of the conformation of apoTaq polymerase in the crystal structure poses a puzzle regarding the mechanisms by which polymerase and 5⬘-nuclease activities are coordinated to generate a nick in the nucleotide replacement reaction (8). The maximum molecular dimension of the crystal structure, which is extended, is 130 Å, with the active sites of the polymerase and nuclease domains 70 Å apart. A study by solution small angle x-ray scattering (SAXS)1 confirms an expanded conformation similar to that in the crystal structure (19). On the other hand, the crystal structure of Taq polymerase bound to an antibody (Fab) inhibitor (20, 21) shows that the Klentaq and 5⬘-nuclease domains can adopt significantly different orientations, and the two active sites are brought significantly closer together, to about 40 Å. However, because the Fab inhibitor does not structurally resemble the DNA substrate, the Taq polymerase-Fab complex structure cannot be used to explain the coordination of the polymerase and the nuclease activities in nick generation. To date, there has been no structural information with Taq polymerase bound to a DNA substrate that is appropriate for an examination of its nick generation function. We have used solution small angle neutron scattering (SANS) to study the conformational changes in Taq polymerase, as well as in individual Klentaq and 5⬘-nuclease domains, upon binding to the overlap flap DNA substrate. By using the contrast variation technique, SANS can study the conformation changes of the Taq polymerase and DNA substrate components separately because the scattering of neutrons from either the DNA or the protein can be contrast matched by that of the buffer in 65 or 42% D2O, respectively (22). In this way, either the DNA or the protein is rendered “invisible” to neutrons. The results show that Taq polymerase adopts a more compact conformation when bound to the DNA substrate. However, the conformation change in Taq polymerase is not enough to bring the active sites on the polymerase and 5⬘-nuclease into close proximity. With recent progress in computational methods for analyzing solution scattering data, a three-dimensional low resolution structure (or “envelope”) of a compact biological macromolecule can be reconstructed from solution small angle scattering profiles (23–27). A three-dimensional molecular envelope of the Taq polymerase-DNA complex reconstructed from SANS data reveals that the 5⬘-nuclease domain adopts an orientation that is different from its orientation in the two known crystal structures of Taq polymerase. Moreover, the upstream and the downstream duplex portions of the DNA substrate in the pro1 The abbreviations used are: SAXS, small angle x-ray scattering; P(r), length distribution function; Rg, radius of gyration; SANS, small angle neutron scattering.

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tein-DNA complex form an angle of about 120° with respect to each other. Our SANS results thus support the footprinting results from Taq polymerase (11) and the E. coli polymerase I (2) which find that the polymerase domain and the 5⬘-nuclease domain can operate independently but collaborate with each other to some extent. EXPERIMENTAL PROCEDURES

Protein Expression and Purification—The recombinant expression plasmid for Taq polymerase was constructed by inserting the gene of Taq DNA polymerase (amino acid codons 1– 832) into the pET-22b vector (Novagen). The expression plasmids for individual Klentaq and Taq 5⬘-nuclease proteins were constructed into the pET-28c vector (Novagen), which has a sequence encoding a C-terminal His6 tag. All three plasmids were transformed into E. coli strain BL21(DE3). The cells containing the plasmids were grown at 37 °C in TB medium containing 80 mg/liter ampicillin to an A600 of about 1.0. The cells were then induced by adding isopropyl-1-thio-␤-D-galactopyranoside to 125 mg/liter and then grown for another 2.5 h and harvested. The harvested cell pellet was resuspended in 25 ml/liter culture of buffer A containing 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 100 mM NaCl, and 4 mg/ml lysozyme/liter of original culture. After the resuspended mixture was incubated at room temperature for 15 min, phenylmethylsulfonyl fluoride was added to 0.1 mM. The mixture was then sonicated to break open the cells and centrifuged. The resulting lysate was then heat treated at 75 °C for 40 min and then chilled on ice for about 1 h. Phenylmethylsulfonyl fluoride was added again to 0.1 mM, and the resulting mixture was centrifuged at 25,000 ⫻ g for about 20 min. The clarified supernatant containing Taq polymerase was transferred to a clean flask. The DNA was precipitated with the addition polyethylenimine to about 0.25%, and the DNA precipitate was removed by centrifugation at 25,000 ⫻ g. The remaining protein was precipitated from the solution by adding 30 g of ammonium sulfate powder and centrifuged at 25,000 ⫻ g. The protein pellet was resuspended in 10 ml of buffer B containing 25 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, and 20 mM NaCl. The resuspended protein was dialyzed against at least three changes of a large volume of buffer of buffer B containing 0.1 mM phenylmethylsulfonyl fluoride. The solution containing recombinant Taq polymerase was loaded onto a DEAE fast protein liquid chromatography column and eluted with a NaCl gradient ranging from 0 to 300 mM. Taq polymerase eluted at about 140 mM NaCl. The protein was then loaded on a heparin column and eluted at about 320 mM NaCl using a gradient of 0 –1 M NaCl. Klentaq and Taq 5⬘-nuclease domain proteins were purified from a nickel column using the protocols suggested by the manufacturer (Amersham Biosciences). Before SANS experiments, the protein solution was purified further by gel filtration using Superdex-200 (Amersham Biosciences). The purity of each of the proteins was higher than 98% as judged from SDS-PAGE with Coomassie staining. The protein solution was also found to be free of nucleic acid based on the UV spectrum and absorbance at 260 and 280 nm. Protein concentrations were determined by the absorbance at 280 nm in 6.0 M guanidinium HCl solution, using the extinction coefficients ⑀278 ⫽ 1.19 cm⫺1 ml mg⫺1, ⑀278 ⫽ 1.14 cm⫺1 ml mg⫺1, and ⑀278 ⫽ 1.26 for Taq polymerase, Klentaq1, and the 5⬘-nuclease, respectively. DNA Oligonucleotides—The DNA oligonucleotides were synthesized by Integrated DNA Technology Inc. (Coralville, IA) and purified by the PAGE method. The sequences of the nucleotides are 5⬘-CTCAGAAAGAAGGCAACTGGACCGAAGGCG-3⬘, 5⬘-GCTATGAACTCCTTCTCCACAAGCGCCTTCGGTCCAGTTGCCTTCTTTCTGAG-3⬘, and 5⬘-CAGGTTTCTTAAGCTTGTGGAGAAGGAGTTCATAGC-3⬘. When assembled together, these three oligonucleotides form an overlap flap structure in which the upstream primer overlaps with the downstream duplex by one nucleotide as shown in Fig. 1. Sample Preparation for SANS Experiments—For apoTaq polymerase, Klentaq, and 5⬘-nuclease, SANS experiments were conducted in 90% D2O buffer containing 75 mM KCl, 25 mM Tris-HCl, pH 8.0, and 1 mM EDTA. For the SANS experiments on protein-DNA complexes, the three oligonucleotides were dissolved at a 1:1:1 molar ratio in 65% D2O (or 42% D2O) buffer containing 25 mM Tris-HCl, pH 8.0, 75 mM KCl, and 1 mM EDTA, and allowed to anneal at 55 °C for 1 h. The annealed oligonucleotides were then added to solutions of Taq polymerase, Klentaq, or 5⬘-nuclease domains, at a molar ratio of 1:1. In the absence of Mg2⫹ and in the presence of EDTA, the 5⬘-nuclease domain can bind to the overlap flap DNA substrate without cleaving the DNA. The proteinDNA complexes were then purified with gel filtration using Superdex200 to remove impurities from oligonucleotide synthesis (Fig. 2). For

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Conformational States in Taq Polymerase

FIG. 1. A, schematic representation of the replication fork. Polymerase III synthesizes the bulk of leading and laggingstrands synthesis, respectively. Taq polymerase fills the gap, and it is responsible for both synthesizing a new DNA strand and removing the RNA primer to generate a ligatable nick. The lines with arrows represent the extended 5⬘ 3 3⬘strand that is complementary to the template, and the dotted lines represent the RNA primers to be cleaved. B, schematic description of the overlap flap DNA substrate. The DNA substrate consists of a bifurcated downstream duplex and an upstream template-primer duplex. The upstream template-primer duplex overlaps the downstream duplex by 1 bp. The 5⬘nuclease preferred cleavage site is indicated by the arrow (10, 11, 17, 18).

SANS experiments in 90 and 65% D2O buffer, about a 0.5-ml sample or the buffer background was loaded into a 2-mm path length quartz banjo cell and then sealed with paraffin. For SANS experiments in 42% D2O, about a 0.3-ml sample or the buffer background was loaded in 1 mm path-length quartz banjo cell. The protein solution or the protein-DNA complex solutions used for SANS experiments contained about 5 mg/ml protein. SANS Experiments and Data Reduction—SANS measurements were conducted on the 30-m SANS instrument at the National Institute of Standards and Technology Center for Neutron Research (28). The incident neutron wavelength was ␭ ⫽ 6 Å with a wavelength resolution of ⌬(␭)/␭ ⫽ 15%. The sample-to-detector distances were set at 13.19 and 1.33 m, respectively, to give a Q range from 0.017 Å⫺1 to 0.45 Å⫺1, where Q ⫽ (4␲/␭)sin(␪/2) (␪ is the scattering angle and ␭ is the wavelength) and is the amplitude of scattering vector. Data acquisition time was about 5 h and 2 h at sample-to-detector distances of 13.19 m and 1.33 m, respectively. The scattering intensity was corrected for background and parasitic scattering, placed on an absolute level using a calibrated secondary standard, and averaged circularly to yield the scattering intensity, I(Q), as a function Q (28). The incoherent background from the buffer background was measured, corrected by the volume fraction displaced by the proteins dissolved, and subtracted from the reduced SANS data. The data points in the Q range from about 0.35 Å⫺1 to 0.42 Å⫺1 were then averaged to yield the estimated incoherent background from the proteins in the solution, which was subtracted from the data as well. The SANS experiments were conducted at temperatures of 25 ⫾ 0.1 oC. SANS Data Analysis—In a dilute, monodisperse solution of a protein or other biological macromolecule, the scattering intensity arises from the intramolecular interference and is characteristic of the size, shape, and the molecular weight of the measured molecule (29 –31). The intensity I(Q) of the scattered neutrons from the nuclei of a macromolecule dissolved in buffer solution can be expressed as I共Q兲 ⫽ 共 ␳ P ⫺ ␳ S兲 2 c

MW 2 ␯៮ P共Q兲 NA p

(Eq. 1)

where (␳p ⫺ ␳s)2 is the square of the neutron scattering length density contrast between the neutron scattering length density of the protein ␳p and that of the buffer ␳s, c is the protein concentration in (mg/ml); Mw is the molecular weight, NA is Avogadro’s number, ␯¯ p is the partial specific volume of the protein in the buffer solution, and

P共Q兲 ⫽

1 n2

冘冘冉 n

n

i⫽1 j⫽1

冊

sin Qrij Qrij

FIG. 2. Gel filtration chromatographs of apoTaq polymerase (solid line), overlap flap DNA (dash dot line), and Taq polymerase-DNA complex (dotted line). The figure indicates the absence of aggregations upon addition of DNA to Taq polymerase. The shift in peak position of the Taq polymerase-DNA complex, compared with the peaks of the Taq polymerase and the DNA, respectively, also indicates the formation of the complex and conformational changes of either DNA and/or the protein within the protein-DNA complex. and conformation of a globular protein and can be obtained from the Guinier approximation 1 ln I共Q兲 ⫽ ln I(0) ⫺ Q2Rg2 3

(Eq. 3)

by linear least squares fitting in the QRg ⱕ 1 region. Inverse Fourier transformation of I(Q) gives the length distribution function P(r), which is the probability of finding two scattering points at a given distance r from each other in the measured macromolecule (29). P共r兲 ⫽

1 2␲2

冕

I共Q兲Qr sin共Qr兲dQ

(Eq. 4)

The radius of gyration can also be determined from P(r) by Equation 5. (Eq. 2)

is the form factor that contains information about the size and shape of the measured molecules. Here, rij is the distance between pairs of scattering nuclei i and j. The radius of gyration Rg is related to the size

冕冕

P共r兲r2dr

R g2 ⫽

2 P共r兲dr

(Eq. 5)

Conformational States in Taq Polymerase The value at which P(r) becomes 0 reflects the maximum molecular dimension Dmax of the macromolecule averaged over all orientations. The shape of the P(r) plot and the value of Dmax can be used to deduce the macromolecular shape and the distance between two domains within the protein. Additionally, P(r) can also be derived from the crystal structure coordinates using Equations 2 and 4. One can directly examine the differences between a solution structure and the crystal structure by examining P(r) functions measured by small angle scattering and that calculated from crystal structure. In this study, P(r) was generated from the SANS data by the GNOM program (32). The scattering curves from the various crystal structures were computed using Equation 2. For a multicomponent system, such as a protein-DNA complex in solution, the scattering intensity is (33) I共Q兲 ⫽ 共 ␳ p ⫺ ␳ s兲 2I p共Q兲 ⫹ 共 ␳ p ⫺ ␳ s兲共 ␳ D ⫺ ␳ s兲I pD共Q兲 ⫹ 共 ␳ D ⫺ ␳ s兲 2I D共Q兲 (Eq. 6) where ␳D is the scattering length density of the DNA, ID(Q) is the scattering from the DNA component, and IpD(Q) is the cross-term reflecting the interference between the scattering amplitudes of the DNA and the protein components. In a SANS experiment, the differences between the scattering length density contrast of a protein and that of a DNA can be employed in contrast match experiments to study the structure of one component in a protein-DNA complex while masking the scattering from the other (22). As shown in Equation 6, the contributions of the protein and DNA components within the complex to the scattering intensity depend on their scattering length density contrasts ␳p ⫺␳s and ␳D ⫺ ␳s. The scattering length density is largely dependent on the hydrogen content of a protein or a DNA molecule and can be either calculated from their atomic compositions or measured experimentally. The scattering length density contrast between the buffer solution and a macromolecule can be changed significantly by utilizing the large difference in magnitude and the opposite sign of the scattering lengths of the isotopes of hydrogen and deuterium. As described in Ref. 22, the contrast between DNA and buffer becomes 0 in 65% D2O buffer solutions, whereas the contrast between the protein and the buffer is still significant (22). Then Equation 6 reduces to Equation 1, and only the scattering from the protein is measured. Likewise, in 42% percent D2O buffer solutions the contrast between the protein and buffer becomes 0, and only the scattering from the DNA molecules is detected. Reconstruction of Three-dimensional Molecular Envelopes from SANS Data—In our study, the low resolution three-dimensional molecular structures of the apo and the DNA-bound forms of Taq polymerase and Klentaq were reconstructed by using the program DAMMIN (26, 34) developed by Svergun and Koch to generate a set of PDB-formatted dummy bead coordinates. DAMMIN employs nonlinear minimization to determine the envelope functions, which are parameterized using spherical harmonics and Monte Carlo-based methods to yield models in the form of finite volume element ensembles. The nonlinear constraints on which this algorithm depends are positivity and compactness in scattering density, both of which can usually be satisfied by a natively folded protein. The reconstructed three-dimensional molecular envelopes created from SANS can be docked to the crystal structures in a quantitative manner using the program package Situs developed by Wriggers et al. (35, 36). Situs utilizes vector quantization with topology-representing networks to display three-dimensional structures. A three-dimensional structure is encoded with a set of quantized “codebook vectors” at characteristic positions in the three-dimensional structure. The codebook vectors can identify gross structural features and provide information about the shape and density distribution of a low resolution structure such as those obtained from SANS or of an atomic high resolution structure such as one obtained from x-ray crystallography. The resulting two sets of codebook vectors enable a precise docking and comparison of the low resolution and high resolution structures. In our analysis, the set of PDB-formatted dummy bead coordinates generated by the program DAMMIN from SANS data was transformed into a three-dimensional molecular envelope by Situs to map the coordinates to the three-dimensional density lattice with a hard sphere kernel-convolution of each dummy bead center. The three-dimensional density molecular envelope constructed in this manner was then compared with the crystal structure using the two sets of quantized codebook vectors and the rigid body molecular docking capability of Situs (35, 36). The calculations were performed on an IBM RS6000 computer.

39149 RESULTS

Fig. 3, A, B, and C, summarize the SANS results on the apo and DNA-bound forms of Taq polymerase, Klentaq, and 5⬘nuclease, respectively. The Guinier plots are linear in the low Q region, indicating the absence of aggregation in the protein solution. Gel filtration experiments conducted on Taq polymerase and Taq polymerase-DNA complex also demonstrated the absence of aggregation upon addition of DNA to Taq polymerase (see Fig. 2). The radii of gyration of the apo and DNA-bound forms of Taq polymerase, Klentaq, and 5⬘-nuclease can be obtained from the linear least square fits of the Guinier plots in the QRg ⱕ 1 region. P(r) functions of Taq polymerase of the apo and the DNA-bound forms of the proteins are shown in Fig. 4. When calculating P(r) functions, we chose the scattering intensity of Q ⱕ 0.20 Å⫺1. By doing so, the constraint of compactness needed for subsequent construction of the three-dimension structures was satisfied. Fig. 5 displays (in blue wire) the three-dimensional molecular envelopes that were constructed using DAMMIN (26) and Situs (36). ApoTaq Polymerase Adopts an Expanded Equilibrium Conformation in Solution—SANS shows that Taq polymerase adopts an elongated conformation in solution, similar to that in the crystal structure (8), although the SANS-measured radius of gyration is slightly larger than that computed from the crystal structure (Table I). The SANS-measured Rg of Taq polymerase agrees with that reported by a solution SAXS study (Table I) (19). The SANS measured Dmax is 125 Å, similar to that calculated from the crystal structure. The SANS solution structure and the crystal structure can be compared in more detail by rigid body molecular docking of the two structures. The docking, shown in Fig. 5A, demonstrates that the SANS-generated three-dimensional molecular envelope faithfully reproduces the molecular shape of Taq polymerase. Within the neutron density map, displayed in blue wire graphic format in Fig. 5A, the fingers, thumb, and palm and the inactive 5⬘ 3 3⬘-exonuclease subdomains of the Klentaq domain, as well as the 5⬘-nuclease domain can all be identified. SANS measures the static structure of a protein in solution. The conformation of Taq polymerase measured by SANS (shown in Fig. 5A) reflects time and ensemble averages of the many conformations that are sampled by the Taq polymerase molecule. Because the crystal structures of Taq polymerase show that the protein has two lobes with the Klentaq and the 5⬘-nuclease joined together by a flexible linker, the 5⬘-nuclease can sample a variety of orientations relative to the Klentaq domain. The result that the equilibrium conformation measured by small angle scattering agrees with the crystal structure is probably because small angle scattering probes the weight average of all of the conformations sampled in solution, weighing more heavily the large conformations of a measured molecule. The SANS-generated three-dimensional molecular envelope of the 5⬘-nuclease (shown in Fig. 5C) reveals its characteristic cleft region of the 5⬘-nuclease domain. Nevertheless, the 5⬘nuclease domain measured by SANS in solution is more expanded than that shown in the crystal structure as judged by P(r) and Rg (see Table I). The more compact form of the 5⬘nuclease domain in the crystal structure may result from the presence of Zn2⫹ ions that are complexed with 5⬘-nuclease. If so, our SANS results are consistent with a SAXS study that shows that metal ion binding can induce a more compact conformation in human flap endonuclease I, which is homologous to the 5⬘-nuclease domain of Taq polymerase (37). As for the whole Taq polymerase, rigid body docking of the three-dimensional molecular envelope of the isolated Klentaq

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FIG. 3. I(Q) versus Q plots (shown in insets) and Guinier plots of (A) apoTaq polymerase (f) and Taq polymerase bound to overlap flap DNA (䡺). The SANS experiments on apoTaq polymerase and apoKlentaq were conducted in about 90% D2O. The experiments on protein-DNA complex were conducted in 65% D2O buffer. The scale of the y axis is arbitrary so that the slopes of Guinier plots of the apoTaq polymerase and that of the DNA-bound form of Taq polymerase can be compared. B, apoKlentaq in 90% D2O buffer (●) and Klentaq bound to overlap flap DNA in 65% D2O buffer (E). C, 5⬘-aponuclease domain (Œ) and 5⬘-nuclease domain (‚) bound to overlap flap DNA measured in 65% D2O buffer.

with the corresponding crystal structure (Fig. 5B) allows identification of the thumb and fingers, the palm, and the inactive 3⬘ 3 5⬘-exonuclease regions. The results demonstrate that in

FIG. 4. P(r) functions of A, apoTaq polymerase (f), overlap flap DNA-bound Taq polymerase (䡺), and Taq polymerase bound to Fab inhibitor (solid line) (calculated from the crystal structure, PDB code 1CMW); B, apoKlentaq domain (●) and overlap flap DNA-bound Klentaq domain (E); C, 5ⴕ-aponuclease domain (Œ) and overlap flap DNA-bound 5ⴕ-nuclease domain (‚).

solution, the Klentaq domain also adopts a slightly more expanded conformation than in the crystal structure. As shown in Table I, the radius of gyration of apoKlentaq in solution is slightly larger than that computed from the crystal structure (calculated from the PDB code 1KTQ). This may indicate the flexible nature of the Klentaq domain while it is undergoing a


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FIG. 5. Three-dimensional molecular envelopes obtained from SANS (in blue wire graphic formats). A–C are the apo forms of: A, Taq polymerase with the Klentaq domain colored in red and green and the 5⬘-nuclease colored in orange; B, Klentaq domain; C, 5⬘-nuclease domain. The molecular envelopes in A–C are docked to their respective crystal structures (whole Taq polymerase, Klentaq, and 5⬘-nuclease are from PDB code 1TAQ). D–F are the corresponding overlap flap DNA-bound forms of Taq polymerase (D), the Klentaq domain (E), and the 5⬘-nuclease domain (F). D shows the reorientation of the 5⬘-nuclease domain that occurs upon DNA substrate binding. The direction of reorientation is indicated by the curved orange arrow. The fingers and thumb subdomains, the palm region, and the nuclease domain are labeled. The envelopes in D–F are not docked with the crystal structures.

TABLE I Summary of radius of gyration of apo- and the flap DNA substrate-bound Taq polymerase, Klentaq1, and 5⬘-nuclease determined by SANS Radius of gyration Proteins

Apo Taq polymerase Taq polymerase bound to flap DNA ApoKlentaq Klentaq bound to flap DNA 5⬘-Nuclease 5⬘-Nuclease bound to flap DNA a

Crystal structurea

SANS P(r) fit

SANS Guinier fit

Å

Å

Å

Å

36.9 (PDB code ITAQ) Not available 26.7 (PDB code 1KTQ) Not available 19.9 Not available

38.2 ⫾ 0.8 33.4 ⫾ 0.5 27.4 ⫾ 0.5 26.3 ⫾ 0.5 24.4 ⫾ 1.0 21.4 ⫾ 1.0

38.0 ⫾ 0.6 32.8 ⫾ 0.8 27.1 ⫾ 0.8 26.6 ⫾ 0.6 23.5 ⫾ 0.5 19.0 ⫾ 1.0

38.7 ⫾ 1.0 Not available 31.2 ⫾ 0.9 Not available Not available Not available

SAXS from Guinier fit (Ref. 19)

Calculated from crystal structure using Equations 2, 4, and 5.

variety of small conformational changes (38 – 40). Such a small motion may be quenched in crystals by crystal lattice packing. Overlap Flap DNA Induces 5⬘-Nuclease Domain Rearrangement in Taq DNA Polymerase—As mentioned under “Experimental Procedures,” in 65% D2O solution, the scattering length contrast between a DNA molecule and the buffer background becomes negligible (22). A SANS experiment conducted on a protein-DNA complex under such buffer conditions will reveal

only the conformational changes occurring in the protein component; the DNA component will become invisible. By performing SANS experiments in 65% D2O, we have observed that upon binding the overlap flap DNA substrate, Taq polymerase adopts a more compact conformation than its apo form. This result is shown in the Guinier plot in Fig. 3A, an in P(r) shown in Fig. 4A with an Rg value of 33.4 ⫾ 0.5 Å (calculated from P(r)) for the substrate-bound form of Taq polymerase (for a

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comparison of Rg values, see Table I), which is significantly smaller than Rg ⫽ 38.2 ⫾ 0.8 Å for apoTaq polymerase. The maximum dimension Dmax of DNA-bound Taq polymerase is 105 Å, smaller than that of apoTaq polymerase with a Dmax ⫽ 125 Å. Moreover, as will be presented below, in addition to these changes that result in a more compact Taq polymerase molecule, there also is a major shift in the position of the 5⬘-nuclease domain relative to the Klentaq domain within the enzyme. SANS shows that the overlap flap DNA substrate-bound form of Taq polymerase adopts a compact conformation that is different in at least two ways from the crystal structure of Taq polymerase bound to an inhibitory Fab (PDB code 1CMW) (20, 21). First of all, overlap flap DNA-bound Taq polymerase is less compact than its Fab-bound Taq polymerase counterpart. In the DNA bound form, the Rg is larger (33.4 ⫾ 0.5 Å versus 28.9 Å; Table I) and so is the maximum dimension Dmax (105 Å versus 85 Å); see Fig. 4A). Second, in the SANS-generated structure, the 5⬘-nuclease domain of DNA-bound Taq polymerase has undergone a large scale spatial rearrangement (relative to the Klentaq domain) that is different from that observed in the crystal structure of the Fab-bound Taq polymerase. The three-dimensional molecular envelope of the DNA-bound form of Taq polymerase (in blue wire graphic format) is shown in Fig. 6C, and, for comparison, the crystal structure of Fab-bound Taq polymerase is shown in Fig. 6B. In Fig. 6C, one can see that in the overlap flap Taq polymerase-DNA complex, the 5⬘-nuclease is lifted up toward the thumb and palm subdomains of the Klentaq domain into a position that is occupied by the Fab inhibitor in the corresponding Taq polymerase-Fab crystal complex. On the other hand, in the Taq polymerase-Fab complex, the 5⬘-nuclease domain is folded back so as to be in contact with the finger and palm subdomains and away from the thumb subdomain (for comparison, see the curved arrows in Fig. 6, B and C). The observed changes in the Rg and in P(r) suggest that there are also conformational changes in the isolated Klentaq domain upon binding to overlap flap DNA (see Table I and Fig. 4B). Fig. 4B shows that there is a more prominent shoulder at r ⬇10 Å in P(r). Nevertheless, the close similarity between the molecular envelopes of the apo and DNA substrate-bound forms of Taq polymerase shown in Fig. 5, B and E, reveals that the overall conformation change in Klentaq is clearly not as dramatic as it is in either Taq polymerase or the isolated 5⬘-nuclease domain (see below). Upon binding to DNA, the size of the isolated 5⬘-nuclease domain becomes smaller, indicating that the DNA substrate induces structural changes (see Table I and Fig. 4C). Our SANS result is consistent with the results of a SAXS study of the structural changes of the human flap endonuclease-1 which shows that conformational changes accompany binding of the overlap flap DNA substrate (37). Furthermore, the asymmetric shape of P(r) in Fig. 4C and the asymmetric three-dimensional molecular envelope in Fig. 5F indicate that the 5⬘-nuclease domain adopts an elongated form upon binding to the overlap flap DNA substrate. The DNA Is Bent by 120° When Bound to 5⬘-Nuclease or Taq Polymerase—We have also conducted both SAXS and contrast match SANS experiments to examine conformational changes in the overlap flap DNA upon binding to Taq polymerase or 5⬘-nuclease. The results from SAXS experiments show that the free form of overlap flap DNA (with sequence and number of bp shown in Fig. 1B) is an elongated molecule with Dmax ⫽ 190 Å; for P(r) function, see Fig. 7A. A calculation using h ⫽ 3.4 Å/bp for a B-DNA of 53 bp should give the length of the DNA of about 180.2 Å. Thus, a comparison of the length of the measured DNA

FIG. 6. Comparison of the reorientation of the 5ⴕ-nuclease domain of Taq polymerase upon binding to the Fab inhibitor (20, 21) and binding to the overlap flap DNA. A, the crystal structure of apoTaq polymerase is shown as a reference (PDB code 1TAQ). The domains of Taq polymerase are colored in the same way as in Fig. 5A. B, the crystal structure of Fab inhibitor bound to Taq polymerase (PDB code 1BGX) with the Fab subunit colored in blue and purple. The directions of reorientation of the 5⬘-nuclease domain (relative to the apoTaq polymerase) are indicated in the orange curved arrow to demonstrate the direction of reorientation of the 5⬘-nuclease domain. C, the molecular envelope of overlap flap DNA-bound form of Taq polymerase with the orange curved arrow showing the direction of the 5⬘-nuclease reorientation.

size with that calculated from a model B-DNA indicates that the DNA is a straight B-form duplex with a flap. In 45% D2O buffer solution the neutron scattering length of the protein matches that of the buffer background, the protein component is “invisible.” One can study conformational changes in the DNA component exclusively of a protein-DNA complex. Fig. 7A shows that P(r) function of the DNA in complex with Taq polymerase is significantly different from that of the free form of overlap flap DNA. Fig. 7B shows the reconstructed three-dimensional molecular envelope of the DNA substrate when bound to Taq polymerase. Although SANS is not able to generate a high resolution structure of the DNA, the


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fulgidus flap endonuclease I in complex with a similar overlap flap DNA substrate (42). DISCUSSION

FIG. 7. A, P(r) functions of overlap flap DNA (measured by SAXS; ⽧) and overlap flap DNA bound to Taq polymerase (〫). B, SANS-generated molecular envelope of overlap flap DNA when bound to Taq polymerase (in both blue wire and yellow bead formats). The DNA substrate is bent by 120° when bound to Taq polymerase as indicated by the thick red lines. The flap single-stranded nucleotide is not shown in the neutron map probably because this single-stranded nucleotide is disordered. C, model of SANS-generated molecular envelope of overlap flap DNA-bound Taq polymerase (in both blue wire and red bead formats) docked with the corresponding envelope of the DNA substrate (in blue wire and yellow bead formats).

molecular envelopes in Fig. 7B nonetheless show that the DNA is bent in the Taq polymerase-DNA complex, forming an angle of about 120° presumably at the junction between the upstream duplex and downstream duplex. Such upstream and downstream duplexes bending was also observed in P. furiousus flap endonuclease I interactions with DNA substrates by molecular modeling (41), and in the crystal structure of Archaeoglobus

Our SANS results suggest that, when Taq polymerase binds the overlap flap DNA substrate, tethered Klentaq and 5⬘-nuclease domains and their accompanying active sites do not approach closely enough to allow the simultaneous upstream primer extension and downstream primer cleavage, as speculated, to generate a ligatable nick. These SANS results are similar to those of a footprinting study of in E. coli polymerase I which shows that its polymerase and 5⬘-nuclease domains cannot simultaneously bind to a DNA substrate; they appear to be physically separate and operate independently (2). Nevertheless, our results also show that the overlap flap DNA substrate can induce a change in the orientation of the 5⬘-nuclease domain relative to the Klentaq domain in the intact enzyme. The neutron map shows that the 5⬘-nuclease domain is lifted up and comes in contact with the thumb subdomain and the palm region of the Klentaq domain. The closer contact between the Klentaq and nuclease domains likely explains the results of a site-directed mutagenesis study on Taq polymerase, which suggest that the polymerase and the nuclease domains can influence each other (13–15). In this study, mutations in two groups of amino acid residues, one group in the palm (W417L and G418K), and another group in the thumb subdomain (E507Q), increase the rate of the RNA-dependent 5⬘-nuclease reaction (18). Moreover, a study by the Joyce group (2) has also shown that in intact E. coli DNA polymerase I, the polymerase and 5⬘-nuclease activities are coupled. The bending between the upstream and downstream of the overlap flap DNA observed here may allow the DNA substrate to have an easy access to the active site of the 5⬘-nuclease. The bending of the DNA may also ready the DNA into the right juxtaposition so that the single-stranded 5⬘-flap can thread through the arch region of the 5⬘-nuclease, which was originally suggested by Kim et al. (8). After cleavage of the singlestranded 5⬘-arm, the bending between the upstream and downstream duplexes (or the dynamic fluctuation nature of the DNA nick) may also provide the DNA ligase with a specific DNA substrate to recognize the nick for ligation reaction. Based on ours SANS study, we propose the following functional model for the nick generation process of Taq polymerase which is consistent with footprinting study of E. coli polymerase I (2). The lagging strand synthesis is carried out by the polymerase III. To seal a gap on a DNA duplex, the Klentaq domain binds preferentially to the upstream duplex region of the DNA substrate, upon which it subsequently performs primer extension. When the primer extension reaction reaches the downstream duplex, an additional base is added to cause an overlap with the downstream duplex. This overlap flap structure then becomes the preferred substrate for the 5⬘-nuclease domain, which then binds it and cleaves it at the junction shown in Fig. 1. It is precisely the different preferences of the two domains for different structural features (upstream duplex region versus overlap flap structure) of the structure-specific substrate that ensure that a nick is generated. Further, the closer contact between the Klentaq and nuclease domains may facilitate the transfer of the substrate from the polymerase active site to the nuclease active site. Thus, in a fashion similar to the “facilitated diffusion” seen in other DNA-binding proteins (43), the substrate would be “handed off ” from one active site to the other without much chance of dissociation from the enzyme surface. According to this model, then, Taq polymerase would ensure both the specificity of nick generation and the efficiency of transfer from one active site to the other.

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