Research Article Received: 27 August 2014,
Revised: 30 October 2014,
Accepted: 30 October 2014,
Published online in Wiley Online Library: 26 February 2015
(wileyonlinelibrary.com) DOI: 10.1002/jmr.2446
Solution structure and DNA binding of the catalytic domain of the large serine resolvase TnpX Stephen J. Headeya*, Andrew Sivakumaranb, Vicki Adamsc, Dena Lyrasc, Julian I. Roodc, Martin J. Scanlona and Matthew C. J. Wilceb The transfer of antibiotic resistance between bacteria is mediated by mobile genetic elements such as plasmids and transposons. TnpX is a member of the large serine recombinase subgroup of site-specific recombinases and is responsible for the excision and insertion of mobile genetic elements that encode chloramphenicol resistance in the pathogens Clostridium perfringens and Clostridium difficile. TnpX consists of three structural domains: domain I contains the catalytic site, whereas domains II and III contain DNA-binding motifs. We have solved the solution structure of residues 1–120 of the catalytic domain I of TnpX. The TnpX catalytic domain shares the same overall fold as other serine recombinases; however, differences are evident in the identity of the proposed hydrogen donor and in the size, amino acid composition, conformation, and dynamics of the TnpX active site loops. To obtain the interaction surface of TnpX1–120, we titrated a DNA oligonucleotide containing the circular intermediate joint attCI recombination site into 15N-labeled TnpX1–120 and observed progressive nuclear magnetic resonance chemical shift perturbations using 15N HSQC spectra. Perturbations were largely confined to a region surrounding the catalytic serine and encompassed residues of the active site loops. Utilizing the perturbation map and the data-driven docking program, HADDOCK, we have generated a model of the DNA interaction complex for the TnpX catalytic domain. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: TnpX; serine recombinase; transposon; chloramphenicol resistance; NMR; solution structure; attCI
INTRODUCTION
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Mobile genetic elements such as transposons, conjugative transposons, and integrons play a critical role in the transmission of virulence and antibiotic resistance genes in numerous microorganisms (Adams et al., 2002; Lyras et al., 2004). The excision and insertion of these mobile elements are mediated by tyrosine or serine site-specific recombinases, which also hold much promise as tools for gene therapy (Karow and Calos, 2011). The serine recombinase or resolvase family encompasses a diverse range of protein members and exhibits an extended spectrum of activity (Smith and Thorpe, 2002), including enzymes responsible for the movement of a range of mobile genetic elements including bacteriophage (Bibb et al., 2005; Ghosh and Grove, 2003; Smith et al., 2004), methicillin resistance elements (staphylococcal cassette chromosome mec) from Staphylococcus aureus (Misiura et al., 2013), tetracycline resistance encoded by Tn5397 in Clostridium difficile (Wang and Mullany, 2000), and chloramphenicol resistance encoded by the Tn4451 family of elements from C. difficile and Clostridium perfringens (Adams et al., 2002; Lyras et al., 2004). The large serine resolvases represent a subgroup within the serine recombinase family and are significantly larger (50–82 kDa) than other serine recombinase proteins (~20 kDa) and catalyze a wider range of reactions (Smith and Thorpe, 2002). TnpX homologs belong to the large serine resolvase subgroup and are responsible for the movement of a family of integrative mobile genetic elements that encode chloramphenicol resistance in the pathogens C. difficile and C. perfringens (Adams et al., 2002).
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Large resolvases consist of an N-terminal catalytic domain and a large C-terminal region. Although the N-terminal domain responsible for catalysis is reasonably conserved in all serine recombinases, the C-terminal region is much more variable and comprises 300–600 amino acid residues in the large serine resolvase subgroup (Smith and Thorpe, 2002). Contained within the C-terminal region are a recombinase domain and a zinc ribbon domain, and these domains, together with their linker region, comprise the high-affinity DNA binding determinants (Rutherford et al., 2013). The 82-kDa TnpX protein catalyzes both the insertion and excision of the Tn4451 family of elements (Lyras et al., 2004; Lyras and Rood, 2000). Although initially, the two site-specific recombination events, excision and insertion, were thought to be biochemically the reverse of each other (Crellin and Rood, 1997), they have * Correspondence to: S. Headey, Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia. E-mail: [email protected] a S. J. Headey, M. J. Scanlon Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, 3052, Australia b A. Sivakumaran, M. C. J. Wilce Department of Biochemistry and Molecular Biology, Clayton, Victoria, 3800, Australia c V. Adams, D. Lyras, J. I. Rood Department of Microbiology, Monash University, Clayton, Victoria, 3800, Australia
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SOLUTION STRUCTURE AND DNA BINDING OF THE CATALYTIC DOMAIN OF TNPX been shown to be distinct because of the differences in target site affinity between TnpX and its separate insertion and excision substrates (Adams et al., 2004). A model explaining the directionality of strand transfer in large serine recombinases between the host (attB) and phage (attP) large serine recombinase binding sites has recently been proposed (Rutherford et al., 2013) based on interpretation of the crystal structures of DNA complexes with γδ serine resolvase (Li et al., 2005) and the C-terminal regions of the L1 integrase (Van Duyne and Rutherford, 2013). Interactions in trans between the opposing C-terminal domain coiled-coil regions are proposed to promote recombination for attB/attP pairings, but for the resultant attL/attR sites, the model proposes interactions in cis between the same coiled-coil regions that inhibit recombination. Most large resolvases utilize a phageencoded directionality factor protein to overcome this inhibition and perform the reverse reaction. However, TnpX lacks any such factor (Lyras et al., 2004) but does contain an additional C-terminal binding region that might play a role in determining directionality (Adams et al., 2006). Although not fully understood, the mechanism of catalysis appears to be conserved in all members of the serine recombinase family of site-specific recombinases and involves a 2-bp staggered break across all four DNA strands and the formation of covalent phosphoserine linkages between the DNA strands and recombinase subunits (Hallet and Sherratt, 1997). Linkage formation is thought to be via general acid–base catalysis, but the identities of the acid and base are yet to be confirmed (Grindley et al., 2006). Insights into the mechanisms of strand transfer have been greatly helped by crystal structures of the 183-residue, prototypical γδ serine resolvase in complex with DNA (Li et al., 2005; Mouw et al., 2008). The catalytic synapse appears to consist of four recombinases on the inside with two DNA crossover sites on the outside. This lends itself to a model of strand exchange via a 180° rotation of one half of the complex relative to the other half. However, it is unclear if the synaptic intermediate captured in the crystal represents a state just after strand cleavage or immediately prior to strand exchange (Grindley et al., 2006). Here, we report the solution structure of the TnpX catalytic domain determined by nuclear magnetic resonance (NMR) spectroscopy. Using NMR chemical shift perturbation mapping and data-driven docking, we have obtained a model of the initial interaction complex of the catalytic domain with its target circular intermediate joint attCI DNA recombination site. The model shows a close similarity in the orientation of the DNA with respect to the catalytic domain compared with the γδ resolvase synaptic intermediate crystal structure (Li et al., 2005) and therefore supports the interpretation of the synaptic intermediate crystal structure as representing a state closer to the initial cleavage state.
MATERIALS AND METHODS Construction of glutathione S-transferase (GST)-TnpX1–120 fusion plasmid
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Isotopically labeled protein expression Constructs with GST-TnpX1–120 inserts were used to transform E. coli C43(DE3) cells, which were then grown on Luria broth plates with ampicillin (100 mg/l) overnight. A single colony was picked and grown in 5-ml Luria broth containing ampicillin (100 mg/l), following which the cells were pelleted by centrifugation and resuspended in 500 ml of minimal medium and grown overnight (1 l of minimal medium contained 6 g Na2HPO4, 3 g KH2PO4, 0.5 g NaCl, 1 g NH4Cl, 2 g glucose, 0.24 g MgSO4, 14.7 mg CaCl2·2H2O, and 100 mg ampicillin). The cells were then pelleted by centrifugation and resuspended in 1.5 l of minimal medium that contained 6.7 g/l 15NH4Cl and 6.7 g/l 13 C-glucose to prepare isotopically labeled proteins. This medium also contained 5 ml/l of a vitamin and trace metal solution containing the following per liter of solution: biotin, 0.2 g; CuSO4·5H2O, 2.0 g; NaI, 0.08 g; MnSO4·H2O, 3.0 g; Na2MoO4·2H2O, 0.2 g; boric acid, 0.02 g; CoCl2·6H2O, 0.5 g; ZnCl2, 7.0 g; FeSO4·7H2O, 22.0 g; and H2SO4, 1 ml. The 1.5-l culture was grown in a 2-l baffled stirred-tank fermenter with temperature, pH, and dissolved oxygen regulation. After an initial phase of 3.5 h at 37°C, the culture had reached an optical density at 600 nm (OD600) of 2.74. The temperature was then reduced to 20°C and protein expression induced by 0.5 mM isopropyl-1-thio-βgalactopyranoside for 8 h, achieving a final OD600 value of 5.0. Cells were harvested by centrifugation and the cell pellet stored at 80°C prior to protein purification. Protein purification and analysis The cells were resuspended in 50 ml of buffer A (50 mM 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid, 100 mM L-arginine, 300 mM NaCl, 5% glycerol, pH 7.0) containing a Complete Protease Inhibitor tablet (Roche). The cells were disrupted in a precooled French press, followed by two rounds of centrifugation at 15 000 g for 20 min. The supernatant fraction was loaded onto 2ml GST beads (Promega) preequilibrated in buffer A. The column was washed in 100 ml of buffer A, and the proteins were eluted using buffer A containing 10 mM reduced S-glutathione. The peak fractions were pooled and concentrated in Amicon Microcon 10 centrifugal microconcentrators. To cleave TnpX1–120 from the GST tag, the fusion protein was incubated with 50 units of thrombin overnight at 4°C. TnpX1–120 was purified by gel filtration chromatography on a Pharmacia source Superdex75 16/60 column and analyzed on sodium dodecyl sulfate PAGE gels. The protein was buffer exchanged to 50 mM Na2HPO4 and 150 mM NaCl, pH 7.0, prior to NMR spectroscopy. NMR spectroscopy NMR experiments were performed in Shigemi tubes containing 0.4 mM 15N,13C-TnpX1–120 in 95% H2O/5% 2H2O with 50 mM Na2HPO4 and 150 mM NaCl, pH 7.0. The following spectra were
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The TnpX1–120 region encompassing the catalytic domain, but without its C-terminal, dimerization-inducing E-helix, was amplified by PCR from a plasmid carrying the relevant TnpX sequence (pJIR1999, (Lucet et al., 2005)), using 5′-CATCACGGATCCATGTCA AGGACTTCAAGAATTAC-3′ as forward and 5′-GAGAGTGAATTCCT AGTTGTTACTGTCAATACTGTT-3′ as reverse primers. The forward and reverse primers had BamHI, and EcoRI restriction sites,
respectively, to enable directional cloning into the GST-fusion vector pGEX4T2. In addition, the reverse primer had a stop codon incorporated upstream of the EcoRI restriction site. PCR amplification was performed using an annealing temperature of 50°C and an extension time of 30 s. PCR products were digested with BamHI and EcoRI, purified using the Qiagen PCR purification system, and cloned into pGEX4T2 digested with BamHI and EcoRI. Sequencing in both directions was carried out to ensure that no errors had been introduced.
S. J. HEADEY ET AL. acquired for assignment and structure determination purposes at 25°C on a 600-MHz Varian INOVA spectrometer using a triple resonance cryogenically cooled probe: 2D 1H,1H NOESY (τm 100 ms), 15N HSQC, 13C-HSQC, HNCACB, HNCO, HN(CA)CO, CBCA(CO)NH, (H)CC(CO)NH TOCSY (τm 12 ms), H(CCCO)NH TOCSY (τm 12 ms), HC(C)H-TOCSY, (H)CCH-TOCSY, (HB)CB (CGCD)HD, (HB)CB(CGCDCE)HE, and a 15N-NOESY-HSQC (τm 100 ms). A 2D 1H,1H-NOESY (τm 100 ms) spectrum was also acquired in 100% D2O. A 3D 13C-NOESY-HSQC (τm 100 ms) was acquired at 800 MHz on a Bruker AVANCE spectrometer fitted with a triple resonance cryogenically cooled probe. Standard pulse sequences were used for data acquisition. The 1H chemical shifts were referenced to the water peak, while the 13C and 15N chemical shifts were referenced by the 13C/1H and 15N/1H gyromagnetic ratios. NMR data were processed in TOPSPIN version 2.0 (Bruker Biospin™). The processed spectra were analyzed using XEASY 1.4 software (Bartels et al., 1995). 1H, 13C, and 15N chemical shift assignments were made for 98% of nonlabile resonances and have been deposited in the BioMagResBank database with accession no. 103616.
Structure determination Automated NOE cross-peak picking and structure determination were performed with the ATNOS/CANDID program (Herrmann et al., 2002) using the CNS (Brunger et al., 1998) simulated annealing algorithm. Initial structures were generated from an extended strand conformation using simulated annealing with torsion angle dynamics for the high temperature and fastcooling stages followed by Cartesian dynamics for a second slow-cooling stage. The ATNOS/CANDID program incorporated 112 dihedral restraints derived from the Cα and Cβ chemical shifts. The lowest energy structure was then used as the starting structure for a second round of structure generation in ATNOS/CANDID using Cartesian dynamics for both the high temperature and cooling phases. During this phase, backbone dihedral restraints generated from TALOS (Shen et al., 2009) were included with a tolerance of at least ±20° from the predicted angles. The structures were then refined in CNS with hydrogen bond restraints added where unique donor and acceptor pairs could be determined by structural convergence. The final structures were refined in a layer of TIP3 water molecules in CNS. The 10 lowest energy structures with no NOE violations >0.3 Å, bond violations >0.05 Å, and no angle, improper, or dihedral violations >5° were selected to represent the solution structure of TnpX1–120.
Steady-state NOE
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A steady-state 1H–15N NOE experiment (Farrow et al., 1994) was performed to assess the dynamics of individual backbone N–H bond vectors. Proton saturation was performed at a carrier frequency centered on the water signal during a 5-s recycle delay. Sweep widths were 1944 Hz in the 15N dimension and 8384 Hz in the 1H dimension, and 1024 (1H) × 160 (15N) complex points were acquired with 40 scans per 15N increment. 13C decoupling was applied during the 15N evolution period. The control experiment was performed in an identical manner but without saturation during the recycle delay. The steady-state 1H–15N NOE of each residue was calculated for each data set as the ratio of the peak height in the spectrum recorded with proton saturation
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to that in the spectrum recorded without proton saturation during the NOE delay period. The error was derived from the signal to noise ratios of the experiments.
DNA titration An HSQC titration experiment was conducted to determine the binding site, affinity, and specificity of the interaction between TnpX1–120 and its attCI recombination site. Complementary DNA oligonucleotides representing the attCI site and a randomized control were purchased (Micromon, Clayton). attCI DNA: 5′-TTGACAAACTCGACCCCGAGGGCTATACTTTAATAGGAC 3′-GAACTGTTTGAGCTGGGGCTCCCGATATGAAATTATCCT Randomized DNA: 5′-CCTTAAATGTAAACTTATTAATTATAAAAGTTTACATTTGGATT 3′-GGAATTTACATTTGAATAATTAATATTTTCAAATGTAAACCTAA The oligonucleotides were dissolved in ultrapure water, combined at a concentration of 2 mM, and denatured for 45 s at 95°C then cooled in air to room temperature to allow annealing. The DNA oligonucleotides were added to 0.5-mM samples of 15 13 N, C-TnpX1–120 in 95% H2O/5% 2H2O with 50 mM Na2HPO4 phosphate and 150 mM NaCl, pH 7.0. For the attCI titration, a 15 N-HSQC spectrum was run at each of the following ratios of DNA : protein 0.14, 0.28, 0.43, 0.57, 0.85, and 1. For the randomized control DNA, 15N-HSQC spectra were run at DNA : protein ratios of 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, and 2. The magnitude of the perturbations was calculated using the formula σ = √(0.04 (Nb Nf)2 + (Hb Hf)2) where Nf and Nb and Hf and Hb are the free and bound frequencies of the amide nitrogen and hydrogen, respectively.
Docking A model of the interaction of TnpX with attCI DNA was generated using the program HADDOCK (Dominguez et al., 2003). The family of the 10 lowest energy NMR structures representing the solution structure of TnpX was used for docking. TnpX residues selected for ambiguous interaction restraints comprised the >50%-surface-exposed residues that were perturbed upon attCI addition, namely, Asp17, Asp19, Leu20, Gly22, Glu23, Ser24, Asn25, Gly56, Val57, Arg89, and Asn113 along with four residues identified by random mutagenesis to compromise DNA excision and insertion: Arg13, Ser15, Ser85, and Arg86 (Adams et al., 2006). The catalytic Ser15–Oγ was constrained to within 2 Å of the 5′-phosphate of attCI Gua18. Residues Ser16– Thr21 of loop 1 showed evidence of increased dynamics in their heteronuclear NOEs and were allowed to be fully flexible during annealing. The attCI B-DNA used for docking was generated using Nucleic Acid Builder software, and dihedral angle restraints (B-form nominal values ±45°) and base-pair hydrogen bond restraints were included during docking to preserve its B-DNA form. Residues Cyt16, Cyt17, Gua18, and Ade19 of attCI were designated as ambiguous interaction restraints. Two rounds of docking were performed with the DNA structures from the first round used as the input DNA for the second round of docking. This procedure is recommended to allow the DNA greater flexibility to conform to the protein’s DNA binding site (van Dijk et al., 2006).
Copyright © 2015 John Wiley & Sons, Ltd.
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SOLUTION STRUCTURE AND DNA BINDING OF THE CATALYTIC DOMAIN OF TNPX
RESULTS Solution structure of TnpX1–120 An abridged TnpX1–120 construct was selected for structure determination by NMR spectroscopy as it lacks the dimerizationinducing E-helix. The solution structure of TnpX1–120 is shown in Figure 1A, and the structure statistics are provided in Table 1. The relatively high number of NOE distance restraints generated from our NOESY spectra by the ATNOS/CANDID program (Herrmann et al., 2002) has resulted in a structure with excellent definition in the folded core of the protein. Excluding the unstructured N-termini and C-termini and the first loop region, the root-mean-square deviation (RMSD) for residues Arg6– Arg13 and Ser26–Asn116 is 0.43 Å for backbone atoms and 1.06 Å for all heavy atoms. The catalytic domain of TnpX1–120 has an αβ structure with a central four-stranded parallel β-sheet and three surrounding αhelices (Figure 2A). Our construct lacks the C-terminal E-helix that is responsible for dimerization. A fifth β-strand typical in other serine resolvases is not well formed at its C-terminus in our structure, probably as a result of the E-helix deletion. The unstructured N-terminus is followed by the β1 strand from Leu7 to the catalytic Ser15. Loop 1 extends from Arg16 to Gly22 and is somewhat disordered in the solution structure. The second loop spanning Asp52 to Arg61 is quite well defined with Phe54 and
Phe59 anchoring the loop via hydrophobic contacts with the highly conserved Arg61 protruding its basic functional group toward the active site in the majority of structures (Figure 2A). Loop 3 connects the β3 strand and the αD helix and contains a single-turn αC helix, Asp83–Leu87 that forms part of the catalytic site. The presence of conserved basic residues Arg13, Arg61, Lys82, Arg86, and Arg89 gives the active site its highly basic character. Structures of the 10 lowest energy conformations of TnpX1–120 have been deposited in the Protein Data Bank (PDB) with accession code 2MHC. Fast-timescale dynamics of TnpX1–120 Backbone 1H–15N heteronuclear NOEs provide information about the dynamics of individual N–H bond vectors, with lower values indicative of faster timescale motions compared with the rest of the molecule, consistent with regions of intrinsic disorder. The NOE values that we measured ranged from 0.57 to 0.94 (Figure 1). Low or negative values were observed for the N-termini and C-termini, consistent with their disordered state in the solution structure. The intermediate values between 0.6 and 0.7 measured for Leu20–Gly22 indicate that this central region of loop 1 is relatively more dynamic and less well ordered than the majority of the protein. In comparison, the NOE values for residues Asp52 to Arg61 and residues Asp83 to Arg89 of relatively well-ordered loops 2 and 3, respectively, are between 0.8 Table 1. Structural statistics for TnpX1–120 Pairwise root-mean-square (residues 6–117)a
deviation
Backbone atoms (Å) Heavy atoms (Å) Nonredundant NOE distance restraints Total Intra (i = j) Sequential (|i j| = 1) Short (1 < |i j| ≤ 5) Long (|i j| >5) Dihedral angle restraints Hydrogen bond restraints (two per bond) Deviations from experimental data NOEs (Å) Dihedrals (°) Deviations from ideal geometry Bonds (Å) Angles (°) Impropers (°) Ramachandran statistics (residues 6–117)b Residues in most favored regions Residues in additional allowed regions Residues in generously allowed regions Residues in disallowed regionsc
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0.65 1.40 1931 (16 per residue) 525 499 406 501 158 94 0.040 ± 0.001 0.333 ± 0.032 0.0045 ± 0.0001 0.619 ± 0.013 0.528 ± 0.018 79.0% 17.3% 2.4% 1.3%
a
Mean pairwise root-mean-square deviation of all backbone heavy atoms was 1.54 Å. b As determined by the program PROCHECK-NMR for all residues present in the native sequence except Gly and Pro. c Residues Ile27, Asn58, and Phe59 had backbone dihedral angles in disallowed regions in a minority of structures.
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Figure 1. Solution structure and fast-timescale dynamics of TnpX1–120. 1 15 (A) Stereo view of TnpX1–120 colored according to the measured H, N1 15 NOE with blue residues having H, N-NOE >0.80 and yellow (0.75 to 0.80), orange (0.70 to 0.75), and red (0.02 ppm. Residues with amides perturbed by more than twice the median were Leu14, Asp17, Asp19, Leu20, Gly22, Glu23, Ser24, Asn25, Gln30, Phe54, Gly56, Val57, Asn58, Met84, Arg89, Phe109, Leu110, Ala111, Asn113, Ile116, and Asp117. The following TnpX1–120 residues showed decreases in their peak heights of >20% upon equimolar addi-
tion of attCI: Leu20, Gly22, Phe54, Gly56, Asn58, Asn60, Asn114, Asp117, and Asn119. These residues form a proposed DNA binding site that surrounds the catalytic triad (Figure 4). An equilibrium dissociation constant could not be derived from the titration data as a consequence of the low affinity of the interaction and the lack of curvature in the dose response, indicating a low-affinity interaction in the millimolar range. The randomized DNA construct was titrated into 15N-labeled TnpX1–120 up to a DNA : protein ratio of 2. Specific backbone amide resonances of TnpX1–120 were perturbed upon the randomized DNA addition, although the number and magnitude of perturbations were much less than that seen for attCI DNA. Only four residues had amide resonances with perturbations >0.02 ppm: Ser15, Asp17, Asp19, and Asn56. The following TnpX1–120 residues showed decreases in their peak heights of >20% upon addition of the randomized DNA at a DNA : protein ratio of 2:1: Asp17, Thr21, Gly22, Asn25, Ile27, Phe54, and Asn119. Although the perturbed residues are localized around the catalytic site, the lower magnitudes of perturbations are consistent with very weak binding to the randomized DNA. Modeling the initial interaction of the recombinase catalytic domain with its DNA target
15
15
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Figure 3. N-HSQC spectra of TnpX1–120. N-HSQC spectra with selec15 13 tive elimination of secondary amide signals of N, C-TnpX 0.5 mM in the apo state (black) and in the presence of 0.5 mM attCI DNA (red).
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Considerable conformational rearrangements of protein and DNA occur during strand transfer. However, it is unclear if the synaptic intermediate captured in the crystal represents a state just after strand cleavage or immediately prior to strand exchange or whether these states differ substantially (Grindley et al., 2006). The NMR perturbation map that we derived from the attCI titration reveals a binding interface that enabled us to use data-driven docking of the TnpX1–120 catalytic domain with the target attCI DNA in order to create a model of their initial interaction state. Using the perturbation map as the basis for ambiguous distance restraints, docking was performed using HADDOCK (Dominguez et al., 2003). One thousand initial structures were rigid body docked. The 200 lowest energy structures were subsequently refined using simulated annealing in a vacuum and then in a layer of TIP3 water molecules. Structures were clustered by RMSD and the clusters sorted by their HADDOCK
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SOLUTION STRUCTURE AND DNA BINDING OF THE CATALYTIC DOMAIN OF TNPX
Figure 4. NMR perturbation map and mutational analysis of TnpX1–120. (A) NMR structure of TnpX1–120 with residues with backbone amides perturbed upon addition of attCI shown in pink. (B) The same structure with residues important to function as identified by random mutagenesis (Adams et al., 2006) shown in yellow. (C) Electrostatic surface representation of TnpX1–120 generated using Pymol.
score as recommended. The lowest energy structure from the lowest energy cluster was selected to represent the TnpX–attCI complex. The HADDOCK docking solution depicts the attCI DNA bound to TnpX1–120 with the negatively charged DNA backbone positioned above the conserved basic residues of the active site. The nonconserved acidic residues of loop 1 probe the major groove, while the minor groove is substantially widened (Figure 5A).
DISCUSSION
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The catalytic mechanism for serine recombinases involves the inline displacement of a DNA hydroxyl with a covalent linkage to Oγ of Ser16. A general acid and a general base are thought to act as the proton donor and acceptor, respectively, although their identities remain unclear (Grindley et al., 2006). The arrangement of putative catalytic residues Arg13, Ser15, Arg61,
Arg86, and Arg89 is largely preserved in TnpX, with Arg13 thought to be the most likely candidate as the proton acceptor (Figure 2A). However, at residue 85, located in the single-turn helix of loop 3, the conserved aspartate, as typified in TP901-1 integrase (Figure 2B), is replaced by a serine. A similar substitution is seen in the crystal structure of φC31 integrase from Streptomyces (Smith et al., 2010; PDB 4BQQ; Figure 2C). The conserved aspartate at this position has been proposed to act as the hydrogen donor for catalysis, and its replacement by serine in these large serine resolvases is somewhat of a surprise. Despite the change, Ser85 remains a key residue, with mutational analysis finding that a Ser85Asn substitution in TnpX abrogates both DNA excision and insertion (Adams et al., 2006). It is conceivable that the closely positioned Asp83 in TnpX and its homolog Asp90 in ϕC31 instead take the role of the hydrogen donor, with the Asp83Gly substitution in TnpX also resulting in a catalytically inactive protein (Adams et al., 2006). However, the range of distances between Asp83 and the catalytic Ser15, which makes the covalent linkage with the attCI DNA, was 9.5 to 13.2 Å in our structures, meaning either that the hydrogen atom would need to be shuttled between them or that a sizable conformational change occurs if Asp83 is indeed the donor. Such a conformational change is feasible given that the heteronuclear NOE values for TnpX loop 1 indicate that it is moderately dynamic. Moreover, the closed conformation seen for the corresponding loop 1 of ϕC31 suggests that such fluctuations can occur. However, it is also possible that TnpX Ser85, and its homologue ϕC31 Ser92, might act as an essential link in the hydrogen transfer given that the double substitution occurs in both proteins. Between members of the serine recombinase family, the amino acid composition, conformation, and dynamics of the loops vary considerably. As per the two other large serine recombinase catalytic domains for which coordinates are available, ϕC31 (Smith et al., 2010) and TP901-1 (Yuan et al., 2008), loop 1 of TnpX is relatively large compared with other serine resolvases (PDB: 3LHF, 3ILX, 3G13, 1GDT, 1ZR4, 3PKZ, and 2R0Q). It is somewhat disordered in our solution structure, and this disorder appears to be intrinsic, with the lower 1H–15N heteronuclear NOE values of loop 1 residues indicating that it is quite dynamic at fast timescales (Figure 1). In contrast, loop 2 is well structured, with the aromatic side chains of Phe54 and Phe59 buried in hydrophobic interactions and the 1H–15N-NOE having similar values to the domain average. NMR relaxation measurements are also available for the γδ resolvase of E. coli, but the loop dynamics are quite the reverse with loop 2 being relatively unstructured and considerably more dynamic than loop 1 (Pan et al., 2001). Loop 3 of TnpX1–120 is a single-turn helix with the 1H–15N-NOE supporting a well-ordered confirmation. This is similar to the single-turn helix seen in the crystal structures of γδ resolvase bound to DNA (Li et al., 2005; Yang and Steitz, 1995) but in contrast to its earlier solution structure where the loop is relatively disordered (Pan et al., 2001). The differences in the conformations of loop 3 between the crystal and NMR structures of γδ resolvase might be because the NMR construct lacked the dimerization-inducing E-helix that forms a contact with the loop 3 of the opposing monomer in the dimeric γδ resolvase crystal structures. Our TnpX1–120 construct also lacks the E-helix and is consequently a monomer in solution. Despite this, loop 3 of TnpX1–120 retains the single-turn helix conformation expected in the native state. A fifth β-strand that precedes the E-helix is typical in other serine resolvases but is not as well formed in our TnpX1–120 solution structure, probably because
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Figure 5. TnpX1–120 docked with attCI and γδ resolvase covalently bound to a res I site analog. (A) HADDOCK docking pose of the interaction of TnpX1– 120 with attCI DNA. The docking was achieved using ambiguous interaction restraints derived from the NMR chemical shift perturbation map of attCI DNA binding to TnpX1–120 (illustrated in pink) along with residues identified by mutational analysis (Adams et al., 2006). The lowest energy conformation from the lowest energy cluster is depicted. (B) The monomer of the X-ray crystal structure of a synaptic intermediate of γδ resolvase covalently bound to a DNA substrate via a phosphoserine linkage (Li et al., 2005; (PDB: 1ZR4). (C) An overlay of the TnpX1–120–attCI DNA complex with panel B. (D) The tetramer of the X-ray crystal structure of the synaptic intermediate of γδ resolvase (PDB: 1ZR4). The complex is proposed to represent a state intermediate between strand cleavage and strand exchange with the dotted line indicating the proposed subunit rotation interface.
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of the E-helix deletion. The differences involve only a few Cterminal residues of the construct that are well removed from the catalytic site. Loops 1 and 2 are removed from the putative dimer interface and do not make contacts with the E-helix in the DNA-bound crystal structures of γδ resolvase (Li et al., 2005; Yang and Steitz, 1995). Consequently, their conformations in our monomeric TnpX1–120 construct are likely to reflect the native state. This is in contrast to several serine recombinase structures in apo states where the E-helix has crystallized in conformations that contact loop regions in such a way that would potentially block DNA interactions, raising the concern that these conformations might be crystallization artifacts (3LHF, 3ILX, and 3LHK). Despite the low sequence conservation, residues from all three loops of TnpX were perturbed upon attCI DNA addition. The previously identified primary DNA binding determinants of TnpX are located outside of the N-terminal catalytic domain, the primary and accessory DNA binding sites being in the putative zinc ribbon and recombinase domains, respectively (Adams et al., 2004; Adams et al., 2006). In contrast, the isolated TnpX catalytic domain lacked an observable interaction with attCI DNA in gel shift assays (Adams et al., 2006). Despite the failure of gel shift assays to detect binding, clear evidence of binding is seen in the 15N-HSQC NMR spectra of TnpX, where specific resonances surrounding the active site were progressively perturbed upon attCI DNA addition (Figure 3). The relatively small magnitudes of the perturbations are not consistent with a conformational change in TnpX1–120 upon attCI binding. Rather, progressive peak displacement indicates that the NMR peaks were in fast exchange on the NMR timescale, a regime commonly observed for relatively low affinity, transient interactions. Indeed, the lack of substantial curvature in peak displacement with increasing attCI
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concentration indicates an interaction in the millimolar range: an affinity range in which the ability of the gel shift assays would struggle to detect binding. It might be argued that the NMR perturbation pattern observed in our truncated construct, which lacks the E-helix and C-terminal region recombinase domain and a zinc ribbon domain, might not accurately reflect the interactions of native TnpX. However, when a randomized DNA control was titrated, only very minor perturbations were observable in the 15N-HSQC spectra of TnpX1–120, and only at the higher 2:1 DNA : protein molecular ratio, from which we conclude that the TnpX catalytic domain has a moderate degree of specificity for the attCI site, supporting the contention that the interaction is biologically relevant and not a mere artifact of the truncation. Nevertheless, as with the other serine recombinase/DNA complexes, all of which utilize truncated or mutated constructs, the interaction pattern needs to be interpreted with some caution as reporting a single state of what are undoubtedly multiple interaction states populated during the strand exchange process. The pattern of perturbations upon attCI binding is localized to a region surrounding the active site and including residues of loops 1 and 2 and the single-turn C helix of loop 3 (Figure 4A). The structure, size, and amino acid composition of the loops vary considerably between different members of the serine recombinase family, and their apparent involvement in DNA binding suggests that they might contribute to the observed DNA binding specificity. An analysis of random TnpX mutations identified the following catalytic domain residues as important for TnpX function: the catalytic Ser15; A helix residues Ser26 and Leu34; loop 2 residues Gly56 and Arg61; Ser66 from B helix; and loop 3 residues Asp83, Ser85, Gly88, and Arg89 (Adams et al., 2006). Setting aside the A and B helix mutations, which were likely to have disrupted these structural elements, the remaining
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SOLUTION STRUCTURE AND DNA BINDING OF THE CATALYTIC DOMAIN OF TNPX point mutations taken together with the NMR perturbation map form a near contiguous binding patch on the surface of TnpX (Figure 4B). The absence of slow exchange peaks and generalized line broadening in NMR spectra of TnpX1–120 in the presence of attCI DNA, along with the lack of covalent complexes in gel shift assays, indicate that isolated TnpX1–120 cannot form a covalent linkage with attCI DNA even at the high concentrations required for NMR and despite clear evidence of an interaction. Our construct lacks the E-helix, which mediates dimerization of the catalytic domains and is likely to be required for catalysis. Structural rearrangements of the E-helix relative to the catalytic domain are also proposed to play a role in the interconversion of the presynaptic and strand exchange states while bound to DNA; activating mutations that destabilize the dimer conformation and stabilize the tetramer conformation of the E helix were required to achieve the crystal structure of the γδ resolvase covalently bound to a DNA res I site analog in a tetrameric synapse (Grindley et al., 2006; Li et al., 2005). Thus, the perturbations that we observed most likely reflect the initial interactions that precede the formation of the covalent DNA–protein bond. To visualize this initial interaction, we docked B-form attCI DNA with the TnpX1–120 family of NMR structures using the residues perturbed by DNA addition as ambiguous interaction restraints (Figure 5A). The model depicts the orientation of the DNA as similar to that seen in the crystal structure of the γδ resolvase covalently linked to res I site DNA (Li et al., 2005; Figure 5B). Indeed, the widening of the minor groove in the TnpX1–120–DNA complex is sufficient to accommodate the E-helix in a similar orientation to that seen in the γδ resolvase– DNA complex. The tetrameric form of the γδ resolvase–DNA complex (Figure 5C) is thought to represent a synaptic intermediate in the strand exchange process, but it is not informative as to whether it represents the state immediately after cleavage or just preceding strand exchange (Grindley et al., 2006). However, the
similarities of our TnpX1–120–attCI interaction complex to the γδ resolvase–DNA crystal structure support the contention that this synaptic intermediate represents a state close to the initial cleavage complex.
CONCLUSIONS The catalytic domain I of the large serine recombinase, TnpX, shares the generalized serine recombinase fold in solution. In the absence of the high-affinity binding sites in TnpX domains II and III, the interaction with the attCI integration site DNA was of low affinity but showed preferential binding. The DNA–TnpX1–120 complex that we generated from our NMR data supports the interpretation of the previously described X-ray structure of the γδ resolvase complex with a res I DNA analog as representing a conformation similar to the initial cleavage state.
COMPETING INTERESTS The authors declare that they have no competing interests.
AUTHORS’ CONTRIBUTIONS SH acquired the NMR data, solved the structure, and performed the DNA titrations and the docking. AS and VA designed the construct and expressed the protein. JIR, DL, MS, and MCJW conceived of the study and assisted in experimental design. All authors contributed to the writing and editing of the manuscript.
Acknowledgements This research was supported by a research funding from the National Health and Medical Research Council (NHMRC). MCJW is an NHMRC Senior Research Fellow.
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J. Mol. Recognit. 2015; 28: 316–324
Revised: 30 October 2014,
Accepted: 30 October 2014,
Published online in Wiley Online Library: 26 February 2015
(wileyonlinelibrary.com) DOI: 10.1002/jmr.2446
Solution structure and DNA binding of the catalytic domain of the large serine resolvase TnpX Stephen J. Headeya*, Andrew Sivakumaranb, Vicki Adamsc, Dena Lyrasc, Julian I. Roodc, Martin J. Scanlona and Matthew C. J. Wilceb The transfer of antibiotic resistance between bacteria is mediated by mobile genetic elements such as plasmids and transposons. TnpX is a member of the large serine recombinase subgroup of site-specific recombinases and is responsible for the excision and insertion of mobile genetic elements that encode chloramphenicol resistance in the pathogens Clostridium perfringens and Clostridium difficile. TnpX consists of three structural domains: domain I contains the catalytic site, whereas domains II and III contain DNA-binding motifs. We have solved the solution structure of residues 1–120 of the catalytic domain I of TnpX. The TnpX catalytic domain shares the same overall fold as other serine recombinases; however, differences are evident in the identity of the proposed hydrogen donor and in the size, amino acid composition, conformation, and dynamics of the TnpX active site loops. To obtain the interaction surface of TnpX1–120, we titrated a DNA oligonucleotide containing the circular intermediate joint attCI recombination site into 15N-labeled TnpX1–120 and observed progressive nuclear magnetic resonance chemical shift perturbations using 15N HSQC spectra. Perturbations were largely confined to a region surrounding the catalytic serine and encompassed residues of the active site loops. Utilizing the perturbation map and the data-driven docking program, HADDOCK, we have generated a model of the DNA interaction complex for the TnpX catalytic domain. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: TnpX; serine recombinase; transposon; chloramphenicol resistance; NMR; solution structure; attCI
INTRODUCTION
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Mobile genetic elements such as transposons, conjugative transposons, and integrons play a critical role in the transmission of virulence and antibiotic resistance genes in numerous microorganisms (Adams et al., 2002; Lyras et al., 2004). The excision and insertion of these mobile elements are mediated by tyrosine or serine site-specific recombinases, which also hold much promise as tools for gene therapy (Karow and Calos, 2011). The serine recombinase or resolvase family encompasses a diverse range of protein members and exhibits an extended spectrum of activity (Smith and Thorpe, 2002), including enzymes responsible for the movement of a range of mobile genetic elements including bacteriophage (Bibb et al., 2005; Ghosh and Grove, 2003; Smith et al., 2004), methicillin resistance elements (staphylococcal cassette chromosome mec) from Staphylococcus aureus (Misiura et al., 2013), tetracycline resistance encoded by Tn5397 in Clostridium difficile (Wang and Mullany, 2000), and chloramphenicol resistance encoded by the Tn4451 family of elements from C. difficile and Clostridium perfringens (Adams et al., 2002; Lyras et al., 2004). The large serine resolvases represent a subgroup within the serine recombinase family and are significantly larger (50–82 kDa) than other serine recombinase proteins (~20 kDa) and catalyze a wider range of reactions (Smith and Thorpe, 2002). TnpX homologs belong to the large serine resolvase subgroup and are responsible for the movement of a family of integrative mobile genetic elements that encode chloramphenicol resistance in the pathogens C. difficile and C. perfringens (Adams et al., 2002).
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Large resolvases consist of an N-terminal catalytic domain and a large C-terminal region. Although the N-terminal domain responsible for catalysis is reasonably conserved in all serine recombinases, the C-terminal region is much more variable and comprises 300–600 amino acid residues in the large serine resolvase subgroup (Smith and Thorpe, 2002). Contained within the C-terminal region are a recombinase domain and a zinc ribbon domain, and these domains, together with their linker region, comprise the high-affinity DNA binding determinants (Rutherford et al., 2013). The 82-kDa TnpX protein catalyzes both the insertion and excision of the Tn4451 family of elements (Lyras et al., 2004; Lyras and Rood, 2000). Although initially, the two site-specific recombination events, excision and insertion, were thought to be biochemically the reverse of each other (Crellin and Rood, 1997), they have * Correspondence to: S. Headey, Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia. E-mail: [email protected] a S. J. Headey, M. J. Scanlon Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, 3052, Australia b A. Sivakumaran, M. C. J. Wilce Department of Biochemistry and Molecular Biology, Clayton, Victoria, 3800, Australia c V. Adams, D. Lyras, J. I. Rood Department of Microbiology, Monash University, Clayton, Victoria, 3800, Australia
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SOLUTION STRUCTURE AND DNA BINDING OF THE CATALYTIC DOMAIN OF TNPX been shown to be distinct because of the differences in target site affinity between TnpX and its separate insertion and excision substrates (Adams et al., 2004). A model explaining the directionality of strand transfer in large serine recombinases between the host (attB) and phage (attP) large serine recombinase binding sites has recently been proposed (Rutherford et al., 2013) based on interpretation of the crystal structures of DNA complexes with γδ serine resolvase (Li et al., 2005) and the C-terminal regions of the L1 integrase (Van Duyne and Rutherford, 2013). Interactions in trans between the opposing C-terminal domain coiled-coil regions are proposed to promote recombination for attB/attP pairings, but for the resultant attL/attR sites, the model proposes interactions in cis between the same coiled-coil regions that inhibit recombination. Most large resolvases utilize a phageencoded directionality factor protein to overcome this inhibition and perform the reverse reaction. However, TnpX lacks any such factor (Lyras et al., 2004) but does contain an additional C-terminal binding region that might play a role in determining directionality (Adams et al., 2006). Although not fully understood, the mechanism of catalysis appears to be conserved in all members of the serine recombinase family of site-specific recombinases and involves a 2-bp staggered break across all four DNA strands and the formation of covalent phosphoserine linkages between the DNA strands and recombinase subunits (Hallet and Sherratt, 1997). Linkage formation is thought to be via general acid–base catalysis, but the identities of the acid and base are yet to be confirmed (Grindley et al., 2006). Insights into the mechanisms of strand transfer have been greatly helped by crystal structures of the 183-residue, prototypical γδ serine resolvase in complex with DNA (Li et al., 2005; Mouw et al., 2008). The catalytic synapse appears to consist of four recombinases on the inside with two DNA crossover sites on the outside. This lends itself to a model of strand exchange via a 180° rotation of one half of the complex relative to the other half. However, it is unclear if the synaptic intermediate captured in the crystal represents a state just after strand cleavage or immediately prior to strand exchange (Grindley et al., 2006). Here, we report the solution structure of the TnpX catalytic domain determined by nuclear magnetic resonance (NMR) spectroscopy. Using NMR chemical shift perturbation mapping and data-driven docking, we have obtained a model of the initial interaction complex of the catalytic domain with its target circular intermediate joint attCI DNA recombination site. The model shows a close similarity in the orientation of the DNA with respect to the catalytic domain compared with the γδ resolvase synaptic intermediate crystal structure (Li et al., 2005) and therefore supports the interpretation of the synaptic intermediate crystal structure as representing a state closer to the initial cleavage state.
MATERIALS AND METHODS Construction of glutathione S-transferase (GST)-TnpX1–120 fusion plasmid
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Isotopically labeled protein expression Constructs with GST-TnpX1–120 inserts were used to transform E. coli C43(DE3) cells, which were then grown on Luria broth plates with ampicillin (100 mg/l) overnight. A single colony was picked and grown in 5-ml Luria broth containing ampicillin (100 mg/l), following which the cells were pelleted by centrifugation and resuspended in 500 ml of minimal medium and grown overnight (1 l of minimal medium contained 6 g Na2HPO4, 3 g KH2PO4, 0.5 g NaCl, 1 g NH4Cl, 2 g glucose, 0.24 g MgSO4, 14.7 mg CaCl2·2H2O, and 100 mg ampicillin). The cells were then pelleted by centrifugation and resuspended in 1.5 l of minimal medium that contained 6.7 g/l 15NH4Cl and 6.7 g/l 13 C-glucose to prepare isotopically labeled proteins. This medium also contained 5 ml/l of a vitamin and trace metal solution containing the following per liter of solution: biotin, 0.2 g; CuSO4·5H2O, 2.0 g; NaI, 0.08 g; MnSO4·H2O, 3.0 g; Na2MoO4·2H2O, 0.2 g; boric acid, 0.02 g; CoCl2·6H2O, 0.5 g; ZnCl2, 7.0 g; FeSO4·7H2O, 22.0 g; and H2SO4, 1 ml. The 1.5-l culture was grown in a 2-l baffled stirred-tank fermenter with temperature, pH, and dissolved oxygen regulation. After an initial phase of 3.5 h at 37°C, the culture had reached an optical density at 600 nm (OD600) of 2.74. The temperature was then reduced to 20°C and protein expression induced by 0.5 mM isopropyl-1-thio-βgalactopyranoside for 8 h, achieving a final OD600 value of 5.0. Cells were harvested by centrifugation and the cell pellet stored at 80°C prior to protein purification. Protein purification and analysis The cells were resuspended in 50 ml of buffer A (50 mM 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid, 100 mM L-arginine, 300 mM NaCl, 5% glycerol, pH 7.0) containing a Complete Protease Inhibitor tablet (Roche). The cells were disrupted in a precooled French press, followed by two rounds of centrifugation at 15 000 g for 20 min. The supernatant fraction was loaded onto 2ml GST beads (Promega) preequilibrated in buffer A. The column was washed in 100 ml of buffer A, and the proteins were eluted using buffer A containing 10 mM reduced S-glutathione. The peak fractions were pooled and concentrated in Amicon Microcon 10 centrifugal microconcentrators. To cleave TnpX1–120 from the GST tag, the fusion protein was incubated with 50 units of thrombin overnight at 4°C. TnpX1–120 was purified by gel filtration chromatography on a Pharmacia source Superdex75 16/60 column and analyzed on sodium dodecyl sulfate PAGE gels. The protein was buffer exchanged to 50 mM Na2HPO4 and 150 mM NaCl, pH 7.0, prior to NMR spectroscopy. NMR spectroscopy NMR experiments were performed in Shigemi tubes containing 0.4 mM 15N,13C-TnpX1–120 in 95% H2O/5% 2H2O with 50 mM Na2HPO4 and 150 mM NaCl, pH 7.0. The following spectra were
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The TnpX1–120 region encompassing the catalytic domain, but without its C-terminal, dimerization-inducing E-helix, was amplified by PCR from a plasmid carrying the relevant TnpX sequence (pJIR1999, (Lucet et al., 2005)), using 5′-CATCACGGATCCATGTCA AGGACTTCAAGAATTAC-3′ as forward and 5′-GAGAGTGAATTCCT AGTTGTTACTGTCAATACTGTT-3′ as reverse primers. The forward and reverse primers had BamHI, and EcoRI restriction sites,
respectively, to enable directional cloning into the GST-fusion vector pGEX4T2. In addition, the reverse primer had a stop codon incorporated upstream of the EcoRI restriction site. PCR amplification was performed using an annealing temperature of 50°C and an extension time of 30 s. PCR products were digested with BamHI and EcoRI, purified using the Qiagen PCR purification system, and cloned into pGEX4T2 digested with BamHI and EcoRI. Sequencing in both directions was carried out to ensure that no errors had been introduced.
S. J. HEADEY ET AL. acquired for assignment and structure determination purposes at 25°C on a 600-MHz Varian INOVA spectrometer using a triple resonance cryogenically cooled probe: 2D 1H,1H NOESY (τm 100 ms), 15N HSQC, 13C-HSQC, HNCACB, HNCO, HN(CA)CO, CBCA(CO)NH, (H)CC(CO)NH TOCSY (τm 12 ms), H(CCCO)NH TOCSY (τm 12 ms), HC(C)H-TOCSY, (H)CCH-TOCSY, (HB)CB (CGCD)HD, (HB)CB(CGCDCE)HE, and a 15N-NOESY-HSQC (τm 100 ms). A 2D 1H,1H-NOESY (τm 100 ms) spectrum was also acquired in 100% D2O. A 3D 13C-NOESY-HSQC (τm 100 ms) was acquired at 800 MHz on a Bruker AVANCE spectrometer fitted with a triple resonance cryogenically cooled probe. Standard pulse sequences were used for data acquisition. The 1H chemical shifts were referenced to the water peak, while the 13C and 15N chemical shifts were referenced by the 13C/1H and 15N/1H gyromagnetic ratios. NMR data were processed in TOPSPIN version 2.0 (Bruker Biospin™). The processed spectra were analyzed using XEASY 1.4 software (Bartels et al., 1995). 1H, 13C, and 15N chemical shift assignments were made for 98% of nonlabile resonances and have been deposited in the BioMagResBank database with accession no. 103616.
Structure determination Automated NOE cross-peak picking and structure determination were performed with the ATNOS/CANDID program (Herrmann et al., 2002) using the CNS (Brunger et al., 1998) simulated annealing algorithm. Initial structures were generated from an extended strand conformation using simulated annealing with torsion angle dynamics for the high temperature and fastcooling stages followed by Cartesian dynamics for a second slow-cooling stage. The ATNOS/CANDID program incorporated 112 dihedral restraints derived from the Cα and Cβ chemical shifts. The lowest energy structure was then used as the starting structure for a second round of structure generation in ATNOS/CANDID using Cartesian dynamics for both the high temperature and cooling phases. During this phase, backbone dihedral restraints generated from TALOS (Shen et al., 2009) were included with a tolerance of at least ±20° from the predicted angles. The structures were then refined in CNS with hydrogen bond restraints added where unique donor and acceptor pairs could be determined by structural convergence. The final structures were refined in a layer of TIP3 water molecules in CNS. The 10 lowest energy structures with no NOE violations >0.3 Å, bond violations >0.05 Å, and no angle, improper, or dihedral violations >5° were selected to represent the solution structure of TnpX1–120.
Steady-state NOE
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A steady-state 1H–15N NOE experiment (Farrow et al., 1994) was performed to assess the dynamics of individual backbone N–H bond vectors. Proton saturation was performed at a carrier frequency centered on the water signal during a 5-s recycle delay. Sweep widths were 1944 Hz in the 15N dimension and 8384 Hz in the 1H dimension, and 1024 (1H) × 160 (15N) complex points were acquired with 40 scans per 15N increment. 13C decoupling was applied during the 15N evolution period. The control experiment was performed in an identical manner but without saturation during the recycle delay. The steady-state 1H–15N NOE of each residue was calculated for each data set as the ratio of the peak height in the spectrum recorded with proton saturation
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to that in the spectrum recorded without proton saturation during the NOE delay period. The error was derived from the signal to noise ratios of the experiments.
DNA titration An HSQC titration experiment was conducted to determine the binding site, affinity, and specificity of the interaction between TnpX1–120 and its attCI recombination site. Complementary DNA oligonucleotides representing the attCI site and a randomized control were purchased (Micromon, Clayton). attCI DNA: 5′-TTGACAAACTCGACCCCGAGGGCTATACTTTAATAGGAC 3′-GAACTGTTTGAGCTGGGGCTCCCGATATGAAATTATCCT Randomized DNA: 5′-CCTTAAATGTAAACTTATTAATTATAAAAGTTTACATTTGGATT 3′-GGAATTTACATTTGAATAATTAATATTTTCAAATGTAAACCTAA The oligonucleotides were dissolved in ultrapure water, combined at a concentration of 2 mM, and denatured for 45 s at 95°C then cooled in air to room temperature to allow annealing. The DNA oligonucleotides were added to 0.5-mM samples of 15 13 N, C-TnpX1–120 in 95% H2O/5% 2H2O with 50 mM Na2HPO4 phosphate and 150 mM NaCl, pH 7.0. For the attCI titration, a 15 N-HSQC spectrum was run at each of the following ratios of DNA : protein 0.14, 0.28, 0.43, 0.57, 0.85, and 1. For the randomized control DNA, 15N-HSQC spectra were run at DNA : protein ratios of 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, and 2. The magnitude of the perturbations was calculated using the formula σ = √(0.04 (Nb Nf)2 + (Hb Hf)2) where Nf and Nb and Hf and Hb are the free and bound frequencies of the amide nitrogen and hydrogen, respectively.
Docking A model of the interaction of TnpX with attCI DNA was generated using the program HADDOCK (Dominguez et al., 2003). The family of the 10 lowest energy NMR structures representing the solution structure of TnpX was used for docking. TnpX residues selected for ambiguous interaction restraints comprised the >50%-surface-exposed residues that were perturbed upon attCI addition, namely, Asp17, Asp19, Leu20, Gly22, Glu23, Ser24, Asn25, Gly56, Val57, Arg89, and Asn113 along with four residues identified by random mutagenesis to compromise DNA excision and insertion: Arg13, Ser15, Ser85, and Arg86 (Adams et al., 2006). The catalytic Ser15–Oγ was constrained to within 2 Å of the 5′-phosphate of attCI Gua18. Residues Ser16– Thr21 of loop 1 showed evidence of increased dynamics in their heteronuclear NOEs and were allowed to be fully flexible during annealing. The attCI B-DNA used for docking was generated using Nucleic Acid Builder software, and dihedral angle restraints (B-form nominal values ±45°) and base-pair hydrogen bond restraints were included during docking to preserve its B-DNA form. Residues Cyt16, Cyt17, Gua18, and Ade19 of attCI were designated as ambiguous interaction restraints. Two rounds of docking were performed with the DNA structures from the first round used as the input DNA for the second round of docking. This procedure is recommended to allow the DNA greater flexibility to conform to the protein’s DNA binding site (van Dijk et al., 2006).
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SOLUTION STRUCTURE AND DNA BINDING OF THE CATALYTIC DOMAIN OF TNPX
RESULTS Solution structure of TnpX1–120 An abridged TnpX1–120 construct was selected for structure determination by NMR spectroscopy as it lacks the dimerizationinducing E-helix. The solution structure of TnpX1–120 is shown in Figure 1A, and the structure statistics are provided in Table 1. The relatively high number of NOE distance restraints generated from our NOESY spectra by the ATNOS/CANDID program (Herrmann et al., 2002) has resulted in a structure with excellent definition in the folded core of the protein. Excluding the unstructured N-termini and C-termini and the first loop region, the root-mean-square deviation (RMSD) for residues Arg6– Arg13 and Ser26–Asn116 is 0.43 Å for backbone atoms and 1.06 Å for all heavy atoms. The catalytic domain of TnpX1–120 has an αβ structure with a central four-stranded parallel β-sheet and three surrounding αhelices (Figure 2A). Our construct lacks the C-terminal E-helix that is responsible for dimerization. A fifth β-strand typical in other serine resolvases is not well formed at its C-terminus in our structure, probably as a result of the E-helix deletion. The unstructured N-terminus is followed by the β1 strand from Leu7 to the catalytic Ser15. Loop 1 extends from Arg16 to Gly22 and is somewhat disordered in the solution structure. The second loop spanning Asp52 to Arg61 is quite well defined with Phe54 and
Phe59 anchoring the loop via hydrophobic contacts with the highly conserved Arg61 protruding its basic functional group toward the active site in the majority of structures (Figure 2A). Loop 3 connects the β3 strand and the αD helix and contains a single-turn αC helix, Asp83–Leu87 that forms part of the catalytic site. The presence of conserved basic residues Arg13, Arg61, Lys82, Arg86, and Arg89 gives the active site its highly basic character. Structures of the 10 lowest energy conformations of TnpX1–120 have been deposited in the Protein Data Bank (PDB) with accession code 2MHC. Fast-timescale dynamics of TnpX1–120 Backbone 1H–15N heteronuclear NOEs provide information about the dynamics of individual N–H bond vectors, with lower values indicative of faster timescale motions compared with the rest of the molecule, consistent with regions of intrinsic disorder. The NOE values that we measured ranged from 0.57 to 0.94 (Figure 1). Low or negative values were observed for the N-termini and C-termini, consistent with their disordered state in the solution structure. The intermediate values between 0.6 and 0.7 measured for Leu20–Gly22 indicate that this central region of loop 1 is relatively more dynamic and less well ordered than the majority of the protein. In comparison, the NOE values for residues Asp52 to Arg61 and residues Asp83 to Arg89 of relatively well-ordered loops 2 and 3, respectively, are between 0.8 Table 1. Structural statistics for TnpX1–120 Pairwise root-mean-square (residues 6–117)a
deviation
Backbone atoms (Å) Heavy atoms (Å) Nonredundant NOE distance restraints Total Intra (i = j) Sequential (|i j| = 1) Short (1 < |i j| ≤ 5) Long (|i j| >5) Dihedral angle restraints Hydrogen bond restraints (two per bond) Deviations from experimental data NOEs (Å) Dihedrals (°) Deviations from ideal geometry Bonds (Å) Angles (°) Impropers (°) Ramachandran statistics (residues 6–117)b Residues in most favored regions Residues in additional allowed regions Residues in generously allowed regions Residues in disallowed regionsc
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0.65 1.40 1931 (16 per residue) 525 499 406 501 158 94 0.040 ± 0.001 0.333 ± 0.032 0.0045 ± 0.0001 0.619 ± 0.013 0.528 ± 0.018 79.0% 17.3% 2.4% 1.3%
a
Mean pairwise root-mean-square deviation of all backbone heavy atoms was 1.54 Å. b As determined by the program PROCHECK-NMR for all residues present in the native sequence except Gly and Pro. c Residues Ile27, Asn58, and Phe59 had backbone dihedral angles in disallowed regions in a minority of structures.
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Figure 1. Solution structure and fast-timescale dynamics of TnpX1–120. 1 15 (A) Stereo view of TnpX1–120 colored according to the measured H, N1 15 NOE with blue residues having H, N-NOE >0.80 and yellow (0.75 to 0.80), orange (0.70 to 0.75), and red (0.02 ppm. Residues with amides perturbed by more than twice the median were Leu14, Asp17, Asp19, Leu20, Gly22, Glu23, Ser24, Asn25, Gln30, Phe54, Gly56, Val57, Asn58, Met84, Arg89, Phe109, Leu110, Ala111, Asn113, Ile116, and Asp117. The following TnpX1–120 residues showed decreases in their peak heights of >20% upon equimolar addi-
tion of attCI: Leu20, Gly22, Phe54, Gly56, Asn58, Asn60, Asn114, Asp117, and Asn119. These residues form a proposed DNA binding site that surrounds the catalytic triad (Figure 4). An equilibrium dissociation constant could not be derived from the titration data as a consequence of the low affinity of the interaction and the lack of curvature in the dose response, indicating a low-affinity interaction in the millimolar range. The randomized DNA construct was titrated into 15N-labeled TnpX1–120 up to a DNA : protein ratio of 2. Specific backbone amide resonances of TnpX1–120 were perturbed upon the randomized DNA addition, although the number and magnitude of perturbations were much less than that seen for attCI DNA. Only four residues had amide resonances with perturbations >0.02 ppm: Ser15, Asp17, Asp19, and Asn56. The following TnpX1–120 residues showed decreases in their peak heights of >20% upon addition of the randomized DNA at a DNA : protein ratio of 2:1: Asp17, Thr21, Gly22, Asn25, Ile27, Phe54, and Asn119. Although the perturbed residues are localized around the catalytic site, the lower magnitudes of perturbations are consistent with very weak binding to the randomized DNA. Modeling the initial interaction of the recombinase catalytic domain with its DNA target
15
15
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Figure 3. N-HSQC spectra of TnpX1–120. N-HSQC spectra with selec15 13 tive elimination of secondary amide signals of N, C-TnpX 0.5 mM in the apo state (black) and in the presence of 0.5 mM attCI DNA (red).
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Considerable conformational rearrangements of protein and DNA occur during strand transfer. However, it is unclear if the synaptic intermediate captured in the crystal represents a state just after strand cleavage or immediately prior to strand exchange or whether these states differ substantially (Grindley et al., 2006). The NMR perturbation map that we derived from the attCI titration reveals a binding interface that enabled us to use data-driven docking of the TnpX1–120 catalytic domain with the target attCI DNA in order to create a model of their initial interaction state. Using the perturbation map as the basis for ambiguous distance restraints, docking was performed using HADDOCK (Dominguez et al., 2003). One thousand initial structures were rigid body docked. The 200 lowest energy structures were subsequently refined using simulated annealing in a vacuum and then in a layer of TIP3 water molecules. Structures were clustered by RMSD and the clusters sorted by their HADDOCK
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SOLUTION STRUCTURE AND DNA BINDING OF THE CATALYTIC DOMAIN OF TNPX
Figure 4. NMR perturbation map and mutational analysis of TnpX1–120. (A) NMR structure of TnpX1–120 with residues with backbone amides perturbed upon addition of attCI shown in pink. (B) The same structure with residues important to function as identified by random mutagenesis (Adams et al., 2006) shown in yellow. (C) Electrostatic surface representation of TnpX1–120 generated using Pymol.
score as recommended. The lowest energy structure from the lowest energy cluster was selected to represent the TnpX–attCI complex. The HADDOCK docking solution depicts the attCI DNA bound to TnpX1–120 with the negatively charged DNA backbone positioned above the conserved basic residues of the active site. The nonconserved acidic residues of loop 1 probe the major groove, while the minor groove is substantially widened (Figure 5A).
DISCUSSION
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The catalytic mechanism for serine recombinases involves the inline displacement of a DNA hydroxyl with a covalent linkage to Oγ of Ser16. A general acid and a general base are thought to act as the proton donor and acceptor, respectively, although their identities remain unclear (Grindley et al., 2006). The arrangement of putative catalytic residues Arg13, Ser15, Arg61,
Arg86, and Arg89 is largely preserved in TnpX, with Arg13 thought to be the most likely candidate as the proton acceptor (Figure 2A). However, at residue 85, located in the single-turn helix of loop 3, the conserved aspartate, as typified in TP901-1 integrase (Figure 2B), is replaced by a serine. A similar substitution is seen in the crystal structure of φC31 integrase from Streptomyces (Smith et al., 2010; PDB 4BQQ; Figure 2C). The conserved aspartate at this position has been proposed to act as the hydrogen donor for catalysis, and its replacement by serine in these large serine resolvases is somewhat of a surprise. Despite the change, Ser85 remains a key residue, with mutational analysis finding that a Ser85Asn substitution in TnpX abrogates both DNA excision and insertion (Adams et al., 2006). It is conceivable that the closely positioned Asp83 in TnpX and its homolog Asp90 in ϕC31 instead take the role of the hydrogen donor, with the Asp83Gly substitution in TnpX also resulting in a catalytically inactive protein (Adams et al., 2006). However, the range of distances between Asp83 and the catalytic Ser15, which makes the covalent linkage with the attCI DNA, was 9.5 to 13.2 Å in our structures, meaning either that the hydrogen atom would need to be shuttled between them or that a sizable conformational change occurs if Asp83 is indeed the donor. Such a conformational change is feasible given that the heteronuclear NOE values for TnpX loop 1 indicate that it is moderately dynamic. Moreover, the closed conformation seen for the corresponding loop 1 of ϕC31 suggests that such fluctuations can occur. However, it is also possible that TnpX Ser85, and its homologue ϕC31 Ser92, might act as an essential link in the hydrogen transfer given that the double substitution occurs in both proteins. Between members of the serine recombinase family, the amino acid composition, conformation, and dynamics of the loops vary considerably. As per the two other large serine recombinase catalytic domains for which coordinates are available, ϕC31 (Smith et al., 2010) and TP901-1 (Yuan et al., 2008), loop 1 of TnpX is relatively large compared with other serine resolvases (PDB: 3LHF, 3ILX, 3G13, 1GDT, 1ZR4, 3PKZ, and 2R0Q). It is somewhat disordered in our solution structure, and this disorder appears to be intrinsic, with the lower 1H–15N heteronuclear NOE values of loop 1 residues indicating that it is quite dynamic at fast timescales (Figure 1). In contrast, loop 2 is well structured, with the aromatic side chains of Phe54 and Phe59 buried in hydrophobic interactions and the 1H–15N-NOE having similar values to the domain average. NMR relaxation measurements are also available for the γδ resolvase of E. coli, but the loop dynamics are quite the reverse with loop 2 being relatively unstructured and considerably more dynamic than loop 1 (Pan et al., 2001). Loop 3 of TnpX1–120 is a single-turn helix with the 1H–15N-NOE supporting a well-ordered confirmation. This is similar to the single-turn helix seen in the crystal structures of γδ resolvase bound to DNA (Li et al., 2005; Yang and Steitz, 1995) but in contrast to its earlier solution structure where the loop is relatively disordered (Pan et al., 2001). The differences in the conformations of loop 3 between the crystal and NMR structures of γδ resolvase might be because the NMR construct lacked the dimerization-inducing E-helix that forms a contact with the loop 3 of the opposing monomer in the dimeric γδ resolvase crystal structures. Our TnpX1–120 construct also lacks the E-helix and is consequently a monomer in solution. Despite this, loop 3 of TnpX1–120 retains the single-turn helix conformation expected in the native state. A fifth β-strand that precedes the E-helix is typical in other serine resolvases but is not as well formed in our TnpX1–120 solution structure, probably because
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Figure 5. TnpX1–120 docked with attCI and γδ resolvase covalently bound to a res I site analog. (A) HADDOCK docking pose of the interaction of TnpX1– 120 with attCI DNA. The docking was achieved using ambiguous interaction restraints derived from the NMR chemical shift perturbation map of attCI DNA binding to TnpX1–120 (illustrated in pink) along with residues identified by mutational analysis (Adams et al., 2006). The lowest energy conformation from the lowest energy cluster is depicted. (B) The monomer of the X-ray crystal structure of a synaptic intermediate of γδ resolvase covalently bound to a DNA substrate via a phosphoserine linkage (Li et al., 2005; (PDB: 1ZR4). (C) An overlay of the TnpX1–120–attCI DNA complex with panel B. (D) The tetramer of the X-ray crystal structure of the synaptic intermediate of γδ resolvase (PDB: 1ZR4). The complex is proposed to represent a state intermediate between strand cleavage and strand exchange with the dotted line indicating the proposed subunit rotation interface.
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of the E-helix deletion. The differences involve only a few Cterminal residues of the construct that are well removed from the catalytic site. Loops 1 and 2 are removed from the putative dimer interface and do not make contacts with the E-helix in the DNA-bound crystal structures of γδ resolvase (Li et al., 2005; Yang and Steitz, 1995). Consequently, their conformations in our monomeric TnpX1–120 construct are likely to reflect the native state. This is in contrast to several serine recombinase structures in apo states where the E-helix has crystallized in conformations that contact loop regions in such a way that would potentially block DNA interactions, raising the concern that these conformations might be crystallization artifacts (3LHF, 3ILX, and 3LHK). Despite the low sequence conservation, residues from all three loops of TnpX were perturbed upon attCI DNA addition. The previously identified primary DNA binding determinants of TnpX are located outside of the N-terminal catalytic domain, the primary and accessory DNA binding sites being in the putative zinc ribbon and recombinase domains, respectively (Adams et al., 2004; Adams et al., 2006). In contrast, the isolated TnpX catalytic domain lacked an observable interaction with attCI DNA in gel shift assays (Adams et al., 2006). Despite the failure of gel shift assays to detect binding, clear evidence of binding is seen in the 15N-HSQC NMR spectra of TnpX, where specific resonances surrounding the active site were progressively perturbed upon attCI DNA addition (Figure 3). The relatively small magnitudes of the perturbations are not consistent with a conformational change in TnpX1–120 upon attCI binding. Rather, progressive peak displacement indicates that the NMR peaks were in fast exchange on the NMR timescale, a regime commonly observed for relatively low affinity, transient interactions. Indeed, the lack of substantial curvature in peak displacement with increasing attCI
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concentration indicates an interaction in the millimolar range: an affinity range in which the ability of the gel shift assays would struggle to detect binding. It might be argued that the NMR perturbation pattern observed in our truncated construct, which lacks the E-helix and C-terminal region recombinase domain and a zinc ribbon domain, might not accurately reflect the interactions of native TnpX. However, when a randomized DNA control was titrated, only very minor perturbations were observable in the 15N-HSQC spectra of TnpX1–120, and only at the higher 2:1 DNA : protein molecular ratio, from which we conclude that the TnpX catalytic domain has a moderate degree of specificity for the attCI site, supporting the contention that the interaction is biologically relevant and not a mere artifact of the truncation. Nevertheless, as with the other serine recombinase/DNA complexes, all of which utilize truncated or mutated constructs, the interaction pattern needs to be interpreted with some caution as reporting a single state of what are undoubtedly multiple interaction states populated during the strand exchange process. The pattern of perturbations upon attCI binding is localized to a region surrounding the active site and including residues of loops 1 and 2 and the single-turn C helix of loop 3 (Figure 4A). The structure, size, and amino acid composition of the loops vary considerably between different members of the serine recombinase family, and their apparent involvement in DNA binding suggests that they might contribute to the observed DNA binding specificity. An analysis of random TnpX mutations identified the following catalytic domain residues as important for TnpX function: the catalytic Ser15; A helix residues Ser26 and Leu34; loop 2 residues Gly56 and Arg61; Ser66 from B helix; and loop 3 residues Asp83, Ser85, Gly88, and Arg89 (Adams et al., 2006). Setting aside the A and B helix mutations, which were likely to have disrupted these structural elements, the remaining
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SOLUTION STRUCTURE AND DNA BINDING OF THE CATALYTIC DOMAIN OF TNPX point mutations taken together with the NMR perturbation map form a near contiguous binding patch on the surface of TnpX (Figure 4B). The absence of slow exchange peaks and generalized line broadening in NMR spectra of TnpX1–120 in the presence of attCI DNA, along with the lack of covalent complexes in gel shift assays, indicate that isolated TnpX1–120 cannot form a covalent linkage with attCI DNA even at the high concentrations required for NMR and despite clear evidence of an interaction. Our construct lacks the E-helix, which mediates dimerization of the catalytic domains and is likely to be required for catalysis. Structural rearrangements of the E-helix relative to the catalytic domain are also proposed to play a role in the interconversion of the presynaptic and strand exchange states while bound to DNA; activating mutations that destabilize the dimer conformation and stabilize the tetramer conformation of the E helix were required to achieve the crystal structure of the γδ resolvase covalently bound to a DNA res I site analog in a tetrameric synapse (Grindley et al., 2006; Li et al., 2005). Thus, the perturbations that we observed most likely reflect the initial interactions that precede the formation of the covalent DNA–protein bond. To visualize this initial interaction, we docked B-form attCI DNA with the TnpX1–120 family of NMR structures using the residues perturbed by DNA addition as ambiguous interaction restraints (Figure 5A). The model depicts the orientation of the DNA as similar to that seen in the crystal structure of the γδ resolvase covalently linked to res I site DNA (Li et al., 2005; Figure 5B). Indeed, the widening of the minor groove in the TnpX1–120–DNA complex is sufficient to accommodate the E-helix in a similar orientation to that seen in the γδ resolvase– DNA complex. The tetrameric form of the γδ resolvase–DNA complex (Figure 5C) is thought to represent a synaptic intermediate in the strand exchange process, but it is not informative as to whether it represents the state immediately after cleavage or just preceding strand exchange (Grindley et al., 2006). However, the
similarities of our TnpX1–120–attCI interaction complex to the γδ resolvase–DNA crystal structure support the contention that this synaptic intermediate represents a state close to the initial cleavage complex.
CONCLUSIONS The catalytic domain I of the large serine recombinase, TnpX, shares the generalized serine recombinase fold in solution. In the absence of the high-affinity binding sites in TnpX domains II and III, the interaction with the attCI integration site DNA was of low affinity but showed preferential binding. The DNA–TnpX1–120 complex that we generated from our NMR data supports the interpretation of the previously described X-ray structure of the γδ resolvase complex with a res I DNA analog as representing a conformation similar to the initial cleavage state.
COMPETING INTERESTS The authors declare that they have no competing interests.
AUTHORS’ CONTRIBUTIONS SH acquired the NMR data, solved the structure, and performed the DNA titrations and the docking. AS and VA designed the construct and expressed the protein. JIR, DL, MS, and MCJW conceived of the study and assisted in experimental design. All authors contributed to the writing and editing of the manuscript.
Acknowledgements This research was supported by a research funding from the National Health and Medical Research Council (NHMRC). MCJW is an NHMRC Senior Research Fellow.
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