HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 281, Issue 7, 4261-4266, February 17, 2006

This Article Abstract Full Text (PDF) Supplemental Data All Versions of this Article: 281/7/4261    most recent M511567200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lee, H. H. Articles by Suh, S. W. Search for Related Content PubMed PubMed Citation Articles by Lee, H. H. Articles by Suh, S. W. Social Bookmarking                      What's this?

Crystal Structure of a Metal Ion-bound IS200 Transposase^*

Hyung Ho Lee¹, Ji Young Yoon¹, Hyoun Sook Kim¹, Ji Yong Kang¹, Kyoung Hoon Kim¹, Do Jin Kim¹, Jun Yong Ha¹, Bunzo Mikami, Hye Jin Yoon, and Se Won Suh²

From the Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-742, Korea and Laboratory of Quality Design and Exploitation, Division of Agronomy and Horticultural Science, Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan

Received for publication, October 25, 2005 , and in revised form, November 14, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
IS200 transposases, present in many bacteria and Archaea, appear to be distinct from other groups of transposases. To provide a structural basis for understanding the action of IS200 transposases, we have determined the crystal structure of the SSO1474 protein from Sulfolobus solfataricus, a member of the IS200 family, in both Mn²⁺-bound and Mn²⁺-free forms. Its monomer fold is distinct from other classes of structurally characterized transposases. Two monomers form a tight dimer by exchanging the C-terminal -helix and by merging the two central -sheets into a large -sheet. Glu⁵⁵, His⁶², and four water molecules provide the direct coordination sphere of the catalytically essential metal ion in the Mn²⁺-bound structure. His¹⁶, Asp⁵⁹, and His⁶⁰ also play important roles in maintaining the metal binding site. The catalytic site is formed at the interface between monomers. The candidate nucleophile in the transposition mechanism, strictly conserved Tyr¹²¹ coming from the other monomer, is turned away from the active site, suggesting that a conformational change is likely to occur during the catalytic cycle.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Transposable genetic elements or transposons are mobile DNA sequences that can insert themselves into nonhomologous target sites in the genome. They promote genome rearrangements such as duplication, deletion, and inversion (1). Insertion sequences, the smallest transposable elements, contain a transposase-encoding gene and thus are capable of independent transposition. In most of the insertion sequences, the transposase-encoding gene is flanked by terminal inverted repeats, but some insertion sequences, such as IS200 and IS605, do not carry terminal inverted repeats (2). So far, five protein families have been described that mediate transposition: DDE transposases, Y2 (or rolling circle) transposases, tyrosine transposases, serine transposases, and a fifth family that encodes a combination of reverse transcriptase and endonuclease (RT/En) activities (3). The Mu, Tn5, and DDE transposases, along with retroviral integrases, have been structurally characterized (4–6).

The 707-bp-long insertion sequence IS200, originally identified in Salmonella typhimurium LT2 (7), contains a single open reading frame tnpA, which encodes a unique transposase. Subsequently, IS200-like transposases have been found to be present in a wide variety of Gram-positive and Gram-negative eubacteria and Archaea (8, 9). IS605, IS606, and IS608 from Helicobacter pylori carry two open reading frames, tnpA and tnpB (10). The tnpA open reading frames encode IS200-like transposases. Members of the IS200 transposase family, including that encoded by the H. pylori IS608 tnpA, show high sequence conservation among them, but they do not carry the complete sequence signatures of any of the other known transposases (10). It has also been suggested that the H. pylori insertion sequence IS608 transposes by a mechanism different from that adopted by other known transposases (10).

The crystal structure of an IS200 transposase encoded by the H. pylori IS608 tnpA has very recently been reported (11). It has also been proposed that transposases resembling this H. pylori IS608 transposase be designated as "Y1 transposases," because a single tyrosine residue is absolutely conserved among the family members (11). Mutation of this tyrosine as well as two histidines of the His-hydrophobic-His (or HUH) motif in the H. pylori IS608 transposase leads to loss of the transposase activity, indicating that they are essential for catalysis (11). The HUH motif is suggested to be involved in binding a Mg²⁺ ion (12), which is essential for the catalytic activity of H. pylori IS608 transposase, but its structure does not contain a bound metal ion in the active site (11).

To provide the framework for a better understanding of the unique transposition mechanism of the IS200 transposase family, we have determined the crystal structure of an IS200 transposase encoded by the SSO1474 gene of Sulfolobus solfataricus in both Mn²⁺-bound and Mn²⁺-free forms. S. solfataricus transposase shows 36% sequence identity to the H. pylori IS608 transposase. The structure reveals that the monomer fold of the IS200 transposase family is different from those of other structurally characterized transposase families and that dimerization is necessary for forming the active site at the interface between the monomers. The Mn²⁺-bound structure also reveals a unique coordination sphere for the metal ion. The present structural data shed light on the action of a new class of IS200 transposase family.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Protein Expression and Purification—The SSO1474 gene encoding the 133-residue IS200-like transposase was PCR-amplified and cloned into the expression vector pET-21a(+) (Novagen). This construct was overexpressed in Escherichia coli Rosetta2(DE3)pLysS cells using Terrific-Broth culture medium. Protein expression was induced by 1 mM isopropyl -D-thiogalactopyranoside, and the cells were incubated for an additional 48 h at 18 °C following growth to mid-log phase at 37 °C. The cells were lysed by sonication in 50 mM Tris-HCl (pH 7.0) and 50 mM NaCl. Following heat treatment at 70 °C for 10 min, the sample was centrifuged at 18,000 revolutions/min for 60 min. The supernatant was applied to a HiTrap SP (5 ml) column (Amersham Biosciences), which was previously equilibrated with 50 mM Tris-HCl (pH 7.0). Upon eluting with a gradient of NaCl in the same buffer, SSO1474 transposase was eluted at 650–700 mM NaCl concentration. The protein was further purified by gel filtration on a HiLoad XK-16 Superdex 200 prep-grade column (Amersham Biosciences), which was previously equilibrated with 50 mM Tris-HCl (pH 7.0) and 200 mM NaCl. The procedure for preparing the selenomethionine (SeMet)³-substituted protein was the same, except for the presence of 10 mM dithiothreitol in all buffers used during purification steps. When overexpressing the SeMet-substituted protein in E. coli Rosetta2(DE3)pLysS cells, we used the M9 cell culture medium that contained extra amino acids, including SeMet.

Crystallization—Crystals were grown by the hanging drop vapor diffusion method at 24 °C by mixing equal volumes (2 µl each) of the protein solution (19 mg ml^-1 concentration in 50 mM Tris-HCl (pH 7.0) and 200 mM NaCl) and the reservoir solution. To grow crystals of the native protein, we used a reservoir solution consisting of 1.4 M trisodium citrate and 100 mM sodium HEPES (pH 7.5). The crystals grew to the approximate dimensions of 0.2 x 0.2 x 0.1 mm within a few days. The SeMet-substituted protein was crystallized under crystallization conditions identical to those for the native crystals, except for the presence of 10 mM dithiothreitol in the protein solution. Mn²⁺-bound crystals of the native protein were prepared by replacing the hanging drop solution of the Mn²⁺-free crystals with a solution consisting of 100 mM sodium HEPES (pH 7.5), 25% (w/v) polyethylene glycol 3350, and 100 mM manganese chloride over a period of 30 min in about ten steps before cryoprotection.

X-ray Data Collection and Structure Determination—A crystal of the SeMet-substituted protein was frozen using a cryoprotectant solution containing 10% (v/v) glycerol in the crystallization mother liquor. X-ray diffraction data were collected at 100 K on an Area Detector Systems Corporation (ADSC) Quantum 210 charge-coupled device area detector system at the BL-4A experimental station of the Pohang Light Source, Pohang, Korea. For each image, the crystal was rotated by 1°, and the crystal-to-detector distance was set to 270 mm. The raw data were processed and scaled using the program suite HKL2000 (13). The crystal belongs to the space group C2, with unit cell parameters of a = 93.1 Å, b = 68.2 Å, c = 65.5 Å, = = 90°, = 129.1°. Two monomers are in the asymmetric unit, giving the crystal volume/protein mass (V_m) of 2.52 ų Da^-1 and a solvent content of 51.2%. Table 1 summarizes statistics of multiwavelength anomalous diffraction data collection.

X-ray diffraction data from a native crystal in the Mn²⁺-free form were collected at 100 K on an ADSC Quantum 4R charge-coupled device area detector system at the BL-38B1 experimental station of SPring-8, Harima, Japan. X-ray diffraction data of the Mn²⁺-bound crystal were collected at 100 K on an ADSC Quantum 210 charge-coupled device area detector at the BL-4A experimental station of Pohang Light Source. For cryoprotection of the Mn²⁺-bound crystal, we used a cryoprotectant solution containing 20% (v/v) glycerol in the mother liquor. Table 1 summarizes the statistics of refinement.

View larger version (46K): [in this window] [in a new window]   FIGURE 1. Overall structure of S. solfataricus transposase. A, ribbon diagram of a monomer. -helix and -sheet are in deep salmon and green cyan, respectively. Secondary structure elements were defined by PROMOTIF (25). All of the figures, except Fig. 2, are drawn with PyMOL. B, topology diagram of S. solfataricus transposase. -Strands are shown as arrows and -helices as cylinders. C, ribbon diagram of a dimer. Monomers 1 and 2 are in deep salmon and light blue, respectively. The primed secondary structure elements belong to the second monomer of the dimer. D, stereo C- trace of S. solfataricus transposase. Every tenth residue is marked by a dot and labeled. Two bound manganese (Mn) ions are also indicated. Five signature sequence motifs are highlighted in colored lines: motif I, (Y/V)HUU(W/F)XX(K/R)YRR, at positions 15–25 in purple; motif II, DH(I/V)H(L/I)(L/F)UXXXP, at positions 59–69 in red; motif III, KGXSXR, at positions 81–86 in green; motif IV LWXX(S/G)Y(F/Y)UX(T/S)XG, at positions 100–111 in cyan; and motif V, (I/V)XXYIXXQ, at positions 118–125 in blue, respectively.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Two monomers of the Mn²⁺-free S. solfataricus transposase in the asymmetric unit adopt similar conformations; they overlap with a root mean square (r.m.s.) difference of 0.39 Å for 130 C- atom pairs. Large deviations occur at the residues 111–119, with a maximum of 1.58 Å at Ser¹¹⁴. Two monomers of the Mn²⁺-bound S. solfataricus transposase in the asymmetric unit overlap with an r.m.s. difference of 0.30 Å for 130 C- atom pairs. Large deviations occur at the residues 88–94, with a maximum of 1.14 Å at Pro⁹³. Between the Mn²⁺-free and Mn²⁺-bound monomers, the r.m.s. differences range between 0.27 and 0.48 Å for 130 C- atom pairs. Large deviations occur at the residues 92–98, with a maximum of 2.25 Å at Gly⁹⁸. Unless otherwise stated, we take monomer A for describing the structural features.

View larger version (68K): [in this window] [in a new window]   FIGURE 2. Amino acid sequence alignment of IS200 transposase family members. SSO, S. solfataricus (Swiss Protein Data Base accession code Q97Y68); TM, Thermotoga maritima (Q9X2F2); EC, E. coli (Q8XCI3); HP, H. pylori (O34550); DR, Deinococcus radiodurans (O83028); IS200, S. typhimurium (P59696 [GenBank] ); ISHp608, H. pylori IS608 (Q932W4). Strictly conserved residues and semiconserved residues are colored in pink and green, respectively. Cylinders above the sequences denote -helices and arrows -strands. Blue dotted lines represent disordered residues. Blue circles above the sequence represent positively charged residues that play important roles in DNA binding. Conserved residues of the IS200 transposase family, as indicated in Ref. 10, are marked as green rectangles below the sequence. Red inverted triangles above the sequence indicate the residues that directly coordinate a manganese ion, whereas the strictly conserved Tyr121 is marked with a red rectangle. Five signature sequence motifs are enclosed in colored boxes: motif I (residues 15–25) in purple, motif II (59–69) in red, motif III (81–86) in green, motif IV (100–111) in cyan, and motif V (118–125) in blue, respectively. This figure was drawn with ClustalX (26) and GeneDoc (27).

Comparison of Monomer Structures—The monomer structure of S. solfataricus transposase is similar to that of the H. pylori IS608 transposase (11) (Protein Data Bank (PDB) code 2A6M, sequence identity = 36%, r.m.s. deviation = 1.39 Å for 130 C- atoms of structurally aligned residues of the Mn²⁺-free structure, monomer A). A major difference is the presence of an additional helix at the C terminus of the 155-residue H. pylori transposase. This region is not conserved (Fig. 2), and thus it may be dispensable for the transposition activity.

Comparisons with the structural data base in the Protein Data Bank using the program server DALI (18) found only a low level (Z score 6.5) of structural similarity between S. solfataricus transposase and other functionally unrelated proteins. The two highest Z scores are obtained with a fragment of the E. coli zinc-transporting protein ZntA (46–118), a P1-type ATPase (19) (PDB code 1MWY, Z score = 6.5, sequence identity = 7%, r.m.s. deviation = 2.0 Å for 67 C- atoms of structurally aligned residues) and the fourth metal binding domain from the human Menkes copper-transporting ATPase (20) (PDB code 1AW0 [PDB] , Z score = 6.4, sequence identity = 8%, r.m.s. deviation = 2.0 Å for 66 C- atoms of structurally aligned residues). The structurally similar part corresponds to the core of the S. solfataricus transposase monomer, 2-1-3-4-2-5. This fold resembles the RNA recognition motif or ferredoxin-like fold that is frequently observed in a number of functionally diverse proteins (20, 21).

View larger version (62K): [in this window] [in a new window]   FIGURE 3. A close-up view of a metal ion binding by the HUH motif. A, 2Fo - Fc electron density map around the Mn2+ binding site. Residues near the HUH motif (His60 and His62) are shown in sticks. Octahedral coordination of Mn2+ is represented by black dotted lines, whereas blue dotted lines denote hydrogen bonds. Red balls represent water molecules. B, superposition of the Mn2+-bound (orange sticks) and Mn2+-free (cyan sticks) S. solfataricus transposase structures. Black dotted lines indicate Mn2+ coordination, whereas green dotted lines denote hydrogen bonds. C, superposition of the Mn2+-bound S. solfataricus transposase (orange sticks) and Mn2+-free structure of the H. pylori IS608 transposase (green sticks). Black dotted lines denote Mn2+ coordination and blue dotted lines, hydrogen bonds. D, binding of a divalent metal ion by the HUH motif in other proteins. Black dotted lines denote Mn2+ coordination and hydrogen bonds.

The C-terminal helix 3 is detached from the main body of the monomer and is involved in making contacts with the other monomer in the dimer. It covers the open face of the central -sheet of the second monomer by making nonpolar interactions with the strands 2' and 5' of the neighboring monomer (Fig. 1C). The buried surface area at the interface between monomers of the dimer is 2,100 Ų (protein-protein interaction server at www.biochem.ucl.ac.uk/bsm/PP/server/). Dimerization is essential for the transposition activity, because the active site is formed at the interface between two monomers of the dimer (described under "Mn²⁺ Ion Binding in the Active Site").

Mn²⁺ Ion Binding in the Active Site—Until now, there has been no experimental observation of a catalytically essential Mg²⁺ ion bound to the active site of any IS200 transposase. To provide this missing structural information, we determined the structure of S. solfataricus transposase in the presence of Mn²⁺ ions. In the Mn²⁺-bound structure, an octahedrally coordinated metal ion is bound to each active site of the dimer (Fig. 3A). The distance between the two metal ions in the dimer is 31.2 Å. Thus, the two active sites within a dimer are well separated so that they can bind DNA substrates simultaneously. In both monomers A and B, the manganese ion is hexacoordinated by the side chains of Glu⁵⁵ (located on strand 3), His⁶² (on 4), and four water molecules (Fig. 3A).

In monomer A, the Mn²⁺ ligand distances are 2.5, 2.4, 2.2, 2.4, 2.1, and 2.4 Å for Glu⁵⁵ (O-1 atom), His⁶² (N-1 atom), and the four water molecules (Wat35, Wat32, Wat67, and Wat23), respectively (Fig. 3A). The metal ligand distances were not subject to any restraint during refinement. Making hydrogen bonds, the N-2 atom of the strictly conserved His⁶⁰ holds Wat35 (2.9 Å), and the O-2 atom of Glu⁵⁵ holds Wat32 (2.5 Å). Wat23 is held by hydrogen bonds to the O-1 atom of Glu⁵⁵ and the N-1 atom of His⁶² (3.2 and 3.3 Å, respectively). In monomer B, the Mn²⁺ ligand distances are 2.4, 2.4, 2.5, 2.2, 2.4, and 2.4 Å for Glu⁵⁵ (O-1 atom), His⁶² (N-1 atom), and the four water molecules (Wat9, Wat18, Wat7, and Wat68), respectively. Essentiality of His⁶⁰ and His⁶² in metal ion coordination explains the finding that mutations of the corresponding residues (His⁶⁴ and His⁶⁶) of H. pylori IS608 transposase causes severe reductions in transposition activity (11).

View larger version (66K): [in this window] [in a new window]   FIGURE 4. A surface view of a dimer and DNA binding model of S. solfataricus transposase. A, the electrostatic potential at the molecular surface of a dimer of S. solfataricus transposase is shown (blue, positive; red, negative). B, stem-loop DNA binding to S. solfataricus transposase modeled by superposition of S. solfataricus transposase (Mn2+-free, blue) and the H. pylori IS608 transposase (PDB code 2A6O, orange).

It is hypothesized that the two histidine residues of the HUH motif may be involved in metal ion coordination (12). Our structure of the Mn²⁺-bound S. solfataricus transposase confirmed the proposed role of the HUH motif in the IS200 transposase family. The detailed view of the metal ion binding site provided in this study also has significant implications for a better understanding of the catalytic mechanism of IS200 transposases, because Mg²⁺ ions are essential for DNA nicking and the formation of a covalent intermediate between H. pylori IS608 transposase and DNA (11).

The observed manganese ion binding in the S. solfataricus transposase active site is distinct from the modes of metal ion binding to the HUH motifs of the adeno-associated virus type 5 Rep (AAV Rep), the F plasmid TraI relaxase domain, and the conjugative relaxase TrwC (22–24). In Rep, TraI, and TrwC, two histidines of the HUH motif, along with a third residue located on the preceding -strand (Asp in Rep; His in TraI and TrwC), directly coordinate a zinc, a manganese, and a zinc ion, respectively (Fig. 3D). In comparison, only the second histidine of the HUH motif directly coordinates the metal ion in S. solfataricus transposase, and the first histidine makes an indirect, water-mediated coordination. Our observed structure is also different from the proposed model of the magnesium ion binding to the H. pylori IS608 transposase (11). Our structure indicates that the third Mg²⁺-coordinating ligand in the H. pylori IS608 transposase is most likely Gln⁵⁹, instead of Asp⁶¹ (11) (Fig. 3C).

A significant change in the side chain conformation of Glu⁵⁵ occurs upon Mn²⁺ binding to S. solfataricus transposase (Fig. 3B). The side chain of Glu⁵⁵ changes its orientation so that the terminal oxygen atoms move toward the Mn²⁺ ion. The side chain of His⁶² also undergoes a slight reorientation to coordinate the Mn²⁺ ion. Binding of the metal ion also pulls the side chains of His⁶⁰ and Asp⁵⁹ toward the metal site (Fig. 3B). The extended side chain of Arg²⁵ is located at the dimer interface between the side chains of Asp⁵⁹ and Glu1²³' in the Mn²⁺-free state (supplemental Fig. S1). The primed residue comes from the second monomer of the dimer. However, the side chain of Glu¹²³' changes its direction, and a salt bridge between Arg²⁵ and Glu¹²³' is broken in the Mn²⁺-bound state (supplemental Fig. S1). Glu¹²³' resides on the C-terminal helix 3', and the breakage of a strong salt bridge upon Mn²⁺ binding might have a functional implication. This is because a large conformational change involving the rearrangement of the C-terminal helix 3' appears to be necessary to bring the strictly conserved Tyr¹²¹' into the proximity of the metal ion and its ligands for catalysis (described under "Location of the Strictly Conserved Tyrosine and Implications for Catalyis").

Sequence Motifs and Roles of Conserved Residues—The residues that are highly conserved among the members of the IS200 transposase family can be grouped into five sequence motifs (Fig. 2). Motif I (Y/V)HUU(W/F)XX(K/R)YRR encompasses Tyr¹⁵–Arg²⁵ of S. solfataricus transposase (Fig. 2, boxed in purple). Motif II, DH(I/V)H(L/I)(L/F)UXXXP, corresponds to the HUH motif (12). It covers Asp⁵⁹–Pro⁶⁹ (Fig. 2, boxed in red). Motif III, KGXSXR, covers Lys⁸¹–Arg⁸⁶ (Fig. 2, boxed in green), whereas Motif IV, LWXX(S/G)Y(F/Y)UX(T/S)XG, covers Leu¹⁰⁰–Gly¹¹¹ (Fig. 2, boxed in cyan). Motif V, (I/V)XXYIXXQ, covers Ile¹¹⁸–Gln¹²⁵ (Fig. 2, boxed in blue). It contains the strictly conserved Tyr¹²¹. U is a hydrophobic residue, X stands for any amino acid, and the residues that are strictly conserved in Fig. 2 are in bold face.

Conserved positively charged residues (Lys²²/Arg²⁴ from motif I and Lys⁸¹/Arg⁸⁶ from motif III), along with semiconserved Arg⁷', are clustered on one side of the S. solfataricus dimer (marked with blue circles in Fig. 2 and labeled in Fig. 4A). On the other hand, the opposite face of the dimer is rich in neutral and negatively charged residues (Fig. 4A). The corresponding region of strong positive electrostatic potential has been shown to be the binding site for a stem-loop DNA in the H. pylori IS608 transposase (11). The strictly conserved Lys⁸⁵ and Gly⁸⁶ of the H. pylori IS608 transposase (corresponding to Lys⁸¹ and Gly⁸² of S. solfataricus transposase) have been shown to be crucial for binding the stem-loop DNA (11). We confirmed that S. solfataricus transposase could bind to double-stranded DNA by electrophoretic mobility shift assay (supplemental data and Fig. S2).

When we superimposed the S. solfataricus transposase structure into the stem-loop-bound model of the H. pylori IS608 transposase (11) (PDB code 2A6O), reasonable shape and charge complementarities existed between S. solfataricus transposase and the stem-loop DNA (Fig. 4B). The protruding side chain of Arg⁷ (corresponding to His¹¹ of IS608) penetrated deeply into the major groove, possibly contacting a base pair of the target DNA. In the H. pylori IS608 transposase, His¹¹ interacts with G18 in the major groove (11). Lys²² (corresponding to Lys²⁶ of IS608) neutralizes the negative charge of the phosphate backbone of the stem-loop DNA. The conserved Arg²⁴ (corresponding to Arg²⁸ of IS608) is located near the entrance of the active site and may be essential for interactions with DNA upon possible conformational changes of S. solfataricus transposase (Fig. 4A and supplemental Fig. S3). Arg⁸⁶ (corresponding to Arg⁹⁰ of IS608) is positioned near the apex of the stem-loop, where the DNA backbone makes a turn.

In addition to its role in binding DNA, Lys⁸¹ may also contribute to stabilization of the monomer. In the S. solfataricus transposase structures, the side chain of Lys⁸¹ of motif III points toward the suggested nucleophile Tyr¹²¹' (6.6–7.0 Å between the NZ atom of Lys⁸¹ and the hydroxyl oxygen atom of Tyr¹²¹'). The NZ atom of Lys⁸¹ makes hydrogen bonds with the backbone oxygen atoms of Thr¹⁰² and Ser¹⁰⁴ (2.7–2.8 and 2.8 Å, respectively) (supplemental Fig. S3B). The latter two residues are not conserved. The aliphatic part of the Lys⁸¹ side chain also makes nonpolar interactions with two conserved tryptophan residues (Trp¹⁹ and Trp¹⁰¹) (supplemental Fig. S1).

Strictly conserved Leu¹⁰⁰ and Trp¹⁰¹ from sequence motif IV, together with neighboring residues (Phe¹⁷, Trp¹⁹, Leu⁴⁰, Phe⁸⁰, and Lys⁸¹) contribute to the hydrophobic core of a monomer (supplemental Fig. S1). The conserved Thr¹⁰⁹ from sequence motif IV, located on 5 (Figs. 1D and 2), interacts with the C-terminal helix 3' from the second monomer and appears to contribute to dimerization (Fig. 1D). The side chain OG1 atom of Thr¹⁰⁹ makes a hydrogen bond with the backbone oxygen of Ala¹¹⁰' (2.7 Å). The semiconserved Lys⁴' makes a salt bridge with the semiconserved Glu⁵⁰' at the start of strand 3 (supplemental Fig. S1). This interaction may contribute to stabilizing the N-terminal mini--sheet.

Location of the Strictly Conserved Tyrosine and Implications for Catalysis—Tyr¹²¹ residing on the C-terminal helix 3 is the only tyrosine that is strictly conserved among the IS200 transposase family members (Fig. 2). Mutation of the corresponding residue (Tyr¹²⁷) in the H. pylori IS608 transposase caused loss of the ability to form a covalent intermediate and abolished transposition activity to the background level, whereas mutation of other tyrosines did not significantly affect transposition (11). This result clearly establishes that Tyr¹²¹ acts as the nucleophile in the catalytic mechanism and forms a covalent phosphotyrosine intermediate.

In both our Mn²⁺-free and Mn²⁺-bound structures of S. solfataricus transposase, however, the side chain of Tyr¹²¹' (coming from the second subunit) is turned away from Glu⁵⁵, His⁶⁰, and His⁶², which contribute to formation of the metal binding site (Fig. 3A and supplemental Fig. S1). The distance from the hydroxyl oxygen atom of Tyr¹²¹' to Mn²⁺ is 13.5 Å. Although the precise role of the metal ion in the catalytic mechanism has yet to be determined, formation of the covalent intermediate requires Mg²⁺ (11). This necessitates a conformational change to bring Tyr¹²¹' into the proximity of the metal ion so that the hydroxyl group of Tyr¹²¹' can make a nucleophilic attack on the target DNA. In both DNA-bound and DNA-unbound structures of the H. pylori IS608 transposase, Tyr¹²⁷ is similarly turned away from the HUH motif (11). It has been suggested that an 90° twist of helix D would be required to juxtapose Tyr¹²⁷ of IS608 transposase with the HUH motif (11). The suggested movement may also accompany a change in the side chain orientation of Tyr¹²¹'. This is because the hydroxyl group of Tyr¹²¹' can possibly move a distance of up to a few Å from Mn²⁺ by simply rotating the torsional angle of its side chain. This kind of motion would be made possible if a conformational change in helix 3' removes steric hindrance because of nearby residues, such as Val¹⁸ (supplemental Fig. S1). To confirm such a conformational change involving the C-terminal helix 3', it may be necessary to determine the structure of the metal ion-bound S. solfataricus transposase in complex with the substrate DNA. A possible catalytic mechanism for transposition by S. solfataricus transposase, adapted from Ref. 11, is shown in supplemental Fig. S4, in which the metal ion coordination is based on the present work.

	FOOTNOTES

 
The atomic coordinates and structure factors (code 2F4F and 2F5G) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by a grant from the Korea Ministry of Science and Technology (NRL-2001). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental data and Figs. S1–S4.

¹ Recipients of the BK21 fellowship of the Korean Ministry of Education.

² To whom correspondence should be addressed. Tel.: 82-2-880-6653; Fax: 82-2-889-1568; E-mail: sewonsuh{at}snu.ac.kr.

³ The abbreviations used are: SeMet, selenomethionine; r.m.s., root mean square.

	ACKNOWLEDGMENTS

 
We thank the staff at beamline BL-4A of Pohang Light Source, Pohang, Korea and beamline BL-38B1 of SPring-8, Harima, Japan, for assistance during x-ray data collection.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 

1. Nevers, P., and Saedler, H. (1977) Nature 268, 109-115[CrossRef][Medline] [Order article via Infotrieve]
2. Mahillon, J., and Chandler, M. (1998) Microbiol. Mol. Biol. Rev. 62, 725-774[Abstract/Free Full Text]
3. Curcio, M. J., and Derbyshire, K. M. (2003) Nat. Rev. Mol. Cell Biol. 4, 865-877[CrossRef][Medline] [Order article via Infotrieve]
4. Rice, P., and Mizuuchi, K. (1995) Cell 82, 209-220[CrossRef][Medline] [Order article via Infotrieve]
5. Davies, D. R., Goryshin, I. Y., Reznikoff, W. S., and Rayment, I. (2000) Science 289, 77-85[Abstract/Free Full Text]
6. Dyda, F., Hickman, A. B., Jenkins, T. M., Engelman, A., Craigie, R., and Davies, D. R. (1994) Science 266, 1981-1986[Abstract/Free Full Text]
7. Lam, S., and Roth, J. R. (1983) Cell 34, 951-960[CrossRef][Medline] [Order article via Infotrieve]
8. Devalckenaere, A., Odaert, M., Trieu-Cuot, P., and Simonet, M. (1999) FEMS Microbiol. Lett. 176, 229-233[CrossRef][Medline] [Order article via Infotrieve]
9. Beuzon, C. R., Chessa, D., and Casadesus, J. (2004) Int. Microbiol. 7, 3-12[Medline] [Order article via Infotrieve]
10. Ton-Hoang, B., Guynet, C., Ronning, D. R., Cointin-Marty, B., Dyda, F., and Chandler, M. (2005) EMBO J. 24, 3325-3338[CrossRef][Medline] [Order article via Infotrieve]
11. Ronning, D. R., Guynet, C., Ton-Hoang, B., Perez, Z. N., Ghirlando, R., Chandler, M., and Dyda, F. (2005) Mol. Cell 20, 143-154[CrossRef][Medline] [Order article via Infotrieve]
12. Ilyina, T. V., and Koonin, E. V. (1992) Nucleic Acids Res. 20, 3279-3285[Abstract/Free Full Text]
13. Otwinowski, Z., and Minor, W. (1997) Methods Enzymol. 276, 307-326
14. Terwilliger, T. C., and Berendzen, J. (1999) Acta Crystallogr. Sect. D 55, 849-861[CrossRef][Medline] [Order article via Infotrieve]
15. Terwilliger, T. C. (2000) Acta Crystallogr. Sect. D 56, 965-972[CrossRef][Medline] [Order article via Infotrieve]
16. Jones, T. A., Zou, J. Y., Cowan, S. W., and Kjeldgaard, M. (1991) Acta Crystallogr. Sect. A 47, 110-119[CrossRef]
17. Brünger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Acta Crystallogr. Sect. D 54, 905-921[CrossRef][Medline] [Order article via Infotrieve]
18. Holm, L., and Sander, C. (1993) J. Mol. Biol. 233, 123-138[CrossRef][Medline] [Order article via Infotrieve]
19. Banci, L., Bertini, I., Ciofi-Baffoni, S., Finney, L. A., Outten, C. E., and O'Halloran, T. V. (2002) J. Mol. Biol. 323, 883-897[CrossRef][Medline] [Order article via Infotrieve]
20. Gitschier, J., Moffat, B., Reilly, D., Wood, W. I., and Fairbrother, W. J. (1998) Nat. Struct. Biol. 5, 47-54[CrossRef][Medline] [Order article via Infotrieve]
21. Burd, C. G., and Dreyfuss, G. (1994) Science 265, 615-621[Abstract/Free Full Text]
22. Hickman, A. B., Ronning, D. R., Kotin, R. M., and Dyda, F. (2002) Mol. Cell 10, 327-337[CrossRef][Medline] [Order article via Infotrieve]
23. Datta, S., Larkin, C., and Schildbach, J. F. (2003) Structure 11, 1369-1379[Medline] [Order article via Infotrieve]
24. Guasch, A., Lucas, M., Moncalian, G., Cabezas, M., Perez-Luque, R., Gomis-Ruth, F. X., de la Cruz, F., and Coll, M. (2003) Nat. Struct. Biol. 10, 1002-1010[CrossRef][Medline] [Order article via Infotrieve]
25. Hutchinson, E. G., and Thornton, J. M. (1996) Protein Sci. 5, 212-220[Abstract]
26. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., and Higgins, D. G. (1997) Nucleic Acids Res. 25, 4876-4882[Abstract/Free Full Text]
27. Nicholas, K. B., Nicholas, H. B., Jr., and Deerfield D. W., II (1997) Embnet. News 4, 14

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				J. Filee, P. Siguier, and M. Chandler Insertion Sequence Diversity in Archaea Microbiol. Mol. Biol. Rev., March 1, 2007; 71(1): 121 - 157. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Supplemental Data All Versions of this Article: 281/7/4261    most recent M511567200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lee, H. H. Articles by Suh, S. W. Search for Related Content PubMed PubMed Citation Articles by Lee, H. H. Articles by Suh, S. W. Social Bookmarking                      What's this?