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Structure of the bacteriophage T4 DNA adenine methyltransferase
Abstract
DNA-adenine methylation at certain GATC sites plays a pivotal role in bacterial and phage gene expression as well as bacterial virulence. We report here the crystal structures of the bacteriophage T4Dam DNA adenine methyltransferase (MTase) in a binary complex with the methyl-donor product S-adenosyl-L-homocysteine (AdoHcy) and in a ternary complex with a synthetic 12-bp DNA duplex and AdoHcy. T4Dam contains two domains: a seven-stranded catalytic domain that harbors the binding site for AdoHcy and a DNA binding domain consisting of a five-helix bundle and a β-hairpin that is conserved in the family of GATC-related MTase orthologs. Unexpectedly, the sequence-specific T4Dam bound to DNA in a nonspecific mode that contained two Dam monomers per synthetic duplex, even though the DNA contains a single GATC site. The ternary structure provides a rare snapshot of an enzyme poised for linear diffusion along the DNA.
DNA MTases catalyze methyl group transfer from donor S-adenosyl-L-methionine (AdoMet), producing S-adenosyl-L-homocysteine (AdoHcy) and methylated DNA. Although most prokaryote DNA MTases are components of restriction-modification systems, some MTases are not associated with cognate restriction enzymes, such as the Escherichia coli DNA adenine MTase (Dam). This enzyme methylates an exocyclic amino nitrogen (N6) of the adenine in GATC1,2. Dam MTase gene orthologs are widespread among enteric bacteria and their bacteriophages (see Fig. 1 legend). Dam methylation is not essential for the viability of E. coli, unless it is combined with certain other mutations3; however, Dam is an essential gene in Vibrio cholerae and Yersinia pesudotuberculosis, at least under the tested growth conditions4. Dam methylation is essential for the virulence of Salmonella serovar typhimurium in a murine model of typhoid fever5–7. These observations raise the possibility that Dam inhibitors may have broad antimicrobial action, and dam− mutants of these pathogens may serve as live attenuated vaccines6,8,9.
Structure-based sequence alignment of the selected Dam MTase orthologs. SWISSPROT database accession numbers are listed in parentheses: bacteriophage T4 (P04392) (T4Dam), Escherichia coli (P00475) (EcoDam), and restriction-modification MTases EcoRV (P04393) and DpnIIA (p04043). In addition, the following sequences are used to derive the invariant (white characters highlighted in black) and the conserved residues (light blue): Salmonella typhimurium (P55893), Serratia marcescens (P45454), Yersinia pestis (Q9F7U9), Vibrio cholerae (Q08318), Neisseria meningitides (Q9X3Y7), M.HindIV (P44431), M.MboIA (p34720), M.CviBI (Q01511) and M.MjaIII (Q58015). The secondary structure of T4Dam is indicated above the sequence with cylinders for helices and arrows for strands. The MTase catalytic domain is green, the TRD yellow and the β-hairpin red. The conserved sequence motifs characteristic of DNA-amino MTases are labeled with roman numerals according to Malone et al 21. Dashed lines show the unstructured regions: the loop between strands β6 and β7 is disordered in both the binary and ternary structures of T4Dam, as well as in the M.DpnII-AdoMet structure26. T4Dam was susceptible to proteolysis near residue 250 within this loop (data not shown).
E. coli Dam acts as an efficient de novo and maintenance MTase, methylating both unmethylated and hemimethylated GATC sites with similar efficiency10. The hemimethylated GATC sites, present immediately following DNA replication, regulate the timing and targeting of a number of cellular functions11. In addition, Dam methylation is very important in the E. coli mismatch repair system formed by MutSL and MutH (reviewed in refs. 12,13). Dam methylation also helps to regulate chromosome replication by maintaining the methylation of the origin of chromosome replication. For example, DNA replication is controlled in part by the SeqA protein, which binds specifically to hemimethylated GATC sites near the origin of replication and delays their full methylation14–16. Dam methylation regulates the expression of certain genes in E. coli17,18, the yopE gene in Yersinia pseudotuberculosis19, and phage Mu mom gene (reviewed in ref. 20).
Valuable insights into the organization and function of MTases have come from the characterization of mutations in common motifs discovered by amino acid sequence alignments21. The solution of several MTase crystal structures, M.HhaI22, M.TaqI23, M.HaeIII24, M.PvuII25, M.DpnII26 and M.RsrI27, has contributed essential details related to the specific protein-DNA and protein-cofactor interactions. As a preliminary step to the potential design of Dam inhibitors, we have characterized two structures containing the Dam MTase encoded by bacteriophage T4: one in a binary complex with AdoHcy, and the second in a ternary complex with both AdoHcy and a synthetic 12-bp DNA duplex.
RESULTS
Overall structure of Dam
The monomeric Dam structure contains 9 β-strands and 11 α-helices (Fig. 1). The N-terminal region (residues 1–52) and C-terminal region (residues 149–259) come together, forming the catalytic domain (green in Figs. 1 and and2a)2a) with a seven-stranded β-sheet, a characteristic feature of the class-I AdoMet-dependent MTases28. The N-terminal helices αZ and αA are located on one side of the β-sheet, and helices αC, αD1 and αD2 and αE are on the other side. Between strands β2 and β3 is a separate domain, which we designate as the target-recognition domain (TRD). TRD is composed of a five-helix bundle (αB1–αB5) (yellow in Figs. 1 and and2a)2a) and a 25-residue segment containing a β-hairpin (β8 and β9) inserted between helices αB4 and αB5 (red). The overall dimensions of the molecule are 55 × 34 × 28 Å, with an open cleft located between the catalytic domain (green) and the TRD (yellow and red).
The binary structure of T4Dam–AdoHcy. (a) Ribbon55 representation uses the same color scheme and labels of helices and strands as in Figure 1; the AdoHcy is shown in stick model. (b) Two views of GRASP56 surface representation. The invariant residues (see Fig. 1) are green (catalytic domain), red (β-hairpin) and yellow (helix bundle). T2 and T4Dam differ at three residues57: residues 20 (T4 serine, T2 proline) and 188 (T4 aspartate, T2 glutamate) are colored in blue, residue 26 (T4 asparagine, T2 aspartate) is on a different surface in both views. Single or pair-wise mutations (T4 S20P, T4 N26D or both) converted T4Dam to a T2Damlike enzyme with a higher kcat, whereas T4 D188E showed no change in enzyme activity58. (c) A stereo view of the AdoHcy binding site. The balland- stick representation of AdoHcy is superimposed with a simulated-annealing omit map (Fo – Fc) that contoured at 2.5σ. The hydrogen bonds are shown as dashed lines. (d) Closed pocket (T4Dam) and open pocket (M.DpnII) of the cofactor binding site. The bound AdoHcy and AdoMet are in ball-and-stick model with the sulfur atom in green. The region forms the cover in T4Dam, which includes residues 175–203 (green) after motif IV. The corresponding region in M.DpnII (amino acids 195–212 in red) has a five-residue insertion that is unstructured. (e) A stereo view of the superposition of the active site of T4Dam-AdoHcy (yellow) and M.TaqIDNA (blue) complexes. Two water molecules (red balls) form hydrogenbonds with Asp171 and the backbone carbonyl of Pro172 (wat1) and Tyr181 (wat2). The target adenine-N6 amino group occupies the site of wat1.
Conserved residues and the effects of mutations
Based on the linear arrangement of conserved motifs in DNA MTases, T4Dam is classified in the α group, which has a characteristic motif order of motif I (Phe-X-Gly) followed by TRD and motif IV (Asp-Pro-Pro-Tyr)21. Our structural determination of T4Dam has allowed us to map conserved residues of Dam orthologs and several related restriction-modification MTases onto the structure (Fig. 1). Conserved residues in the primary sequence, including 16 invariant positions, appear scattered throughout the catalytic domain and TRD. The folded structure, however, shows that these invariant residues are clustered on two surface patches (Fig. 2b). We suggest that the conserved surface patches have functional importance, being involved in one of three steps in the methylation reaction: AdoMet binding (Phe32 and Gly34 of motif I, Asp50 of motif II), DNA binding and sequence-specific recognition (Arg91 of αB3, Arg116 and Asn118 of the β-hairpin), and catalysis of methyl transfer (Asp-Pro-Pro-Tyr of motif IV, Lys11 of motif X and Glu254 of β7). The N-terminal Gly9 (motif X) is involved in making a sharp turn that positions the nearby conserved residue Lys11 into the active site (see below).
The structure of the Dam MTase offers a rationale for its conservation. For example, Pro126 (after the β-hairpin) is exposed on the surface together with Arg91, Arg116 and Asn118 (Fig. 2b), invariant residues that have been implicated in specific EcoRV-DNA interactions29. A P126S mutation generates the so-called Damh form of T4Dam and T2Dam30; which is prone to methylate noncognate sites such as the internal adenine in AGACC resulting in resistance to cleavage by the R.EcoP1 restriction endonuclease2. In vivo Damh causes virion DNA hypermethylation31. In vitro T2Damh exhibited a two- to four-fold higher kcat than the wild-type MTase on DNA containing various noncanonical sites; however, the two forms had the same kcat on DNA containing the canonical GATC32. The altered activity of Damh may be due to the involvement of the surface serine hydroxyl in extra hydrogen bonding with DNA at noncognate sites.
Dam-AdoHcy interactions
In the binary Dam–AdoHcy complex, the methyl-donor product AdoHcy (added during purification) is observed at the carboxyl ends of the parallel strands β1, β2 and β4 (Fig. 2a). It is surrounded by the conserved residues from motifs X (Tyr7), I (Phe32-Ser33-Gly34), II (Asp50), VI (Asp171-Pro172-Pro173) and αD1 (Tyr181, Phe184, Trp185) (Fig. 2c). Mutation of Pro172 to alanine or threonine seemed to abolish enzyme activity in vitro, owing to an increase in the Km for AdoMet of 5- or 20-fold, respectively33. Thus, when the AdoMet concentration was raised well above the Km, the overall kcat of these variants was reduced by only two-fold. Pro172 lies next to AdoHcy and the backbone carbonyl oxygen between Asp171 and Pro172 makes van der Waals contact with the homocysteine carbon Cγ (Fig. 2c). The two mutations of Pro172 are likely to change the backbone conformation, thus substantially lowering the affinity for AdoMet and rate of exchange with bound AdoHcy, thereby lowering the kcat.
Dam-AdoHcy interactions can be grouped according to the three moieties of AdoHcy. (i) One side of the adenine ring forms edge-to-face van der Waals contacts with the phenyl ring of Phe32 after strand β1; this interaction is highly conserved among DNA MTases34. On the other side of the adenine ring lies the side chain of Ile51 (after strand β2). The exocyclic amino group (N6) of adenine makes water-mediated hydrogen bonds to the main chain carbonyl oxygen of Phe184. The ring nitrogen N1 of adenine hydrogen bonds to the backbone amide NH of Phe157. (ii) A strongly conserved acidic residue (Asp50) at the carboxyl end of strand β2 forms bifurcated hydrogen bonds with the ribose hydroxyls; this is nearly universal to class-I MTases28. In addition, Tyr7 and Gln52 each form a hydrogen bond with one of the two ribose hydroxyls. (iii) The conserved Gly34 lies underneath the homocysteine moiety, while Asp171 interacts with the amino group (NH3+). The AdoHcy carboxyl oxygens (COO−) interact with the backbone amide NH of Lys11 and the side chain hydroxyl of Ser37.
A unique interaction of T4Dam involves the short helix αD1 (after the Asp-Pro-Pro-Tyr motif), which covers the AdoHcy molecule, nearly burying it in a closed acidic pocket (Fig. 2d). The buried AdoHcy suggests that the exchange between the methyl donor AdoMet and reaction product AdoHcy requires the movement of the 310-helix and the loops flanking it, which may explain the observation that AdoMet is partially co-purified with the enzyme (data not shown). This helix includes Trp185, which is likely to be involved in the quenching of intrinsic tryptophan fluorescence that results from T4Dam binding of either AdoMet or AdoHcy (Kd 10× higher for AdoHcy)35. This suggests that when T4Dam binds its cofactor or methylation product, it undergoes a conformational change that alters the environment of Trp185 (the other two tryptophans, Trp67 and Trp226, are located >20 Å away from the AdoHcy). The same closed-flap AdoHcy binding site is observed in the nonspecific ternary structure (see below); in both cases, the 310-helix and the flanking loops are not involved in any crystal packing contacts. An open conformation was observed in the binary M.DpnII–AdoMet structure26, where the AdoMet is largely visible and the cover (a five-residue longer loop after strand β4) is opened up (Fig. 2d).
Active site
The active site is likely to be situated in a narrow surface pocket next to AdoHcy. The AdoHcy sulfur atom lies in the bottom of the pocket and is visible (Fig. 2d). The loop after strand β4 contributes most of the residues (Asp-Pro-Pro-Tyr of motif IV and Tyr181) in the active site pocket (Fig. 2e). In the absence of the target adenine, the active site is occupied by two ordered water molecules, one of which bridges the side chains of Asp171 and the backbone carbonyl oxygen between Pro172 and Pro173, while the other one interacts with Tyr181. Remarkably, the invariant Asp-Pro-Pro-Tyr of T4Dam is superimposable onto the corresponding motif in the ternary complex of M.TaqI23 (Fig. 2e), as well as the binary complex of M.DpnII26 and M.RsrI27 in the absence of DNA, suggesting that the conformation of Asp-Pro-Pro-Tyr is quite stable and highly conserved. In addition, an invariant Lys11 (motif X) interacts with Asp171 (via charge-charge interaction) and Tyr174 (via hydrogen bond) (Fig. 2e). The Asp171-Lys11-Tyr174 interactions confer additional stability to the active site. Interestingly, the mutant of the corresponding lysine in M.EcoRV (K16R) showed an altered specificity toward the target base36.
The structure of a ternary complex of M.TaqI with DNA and a nonreactive AdoMet analog revealed that the target adenine is swung completely out of the helix and into the active site pocket by torsional rotation of its flanking sugar-phosphate backbone bonds23, so called ‘base flipping’22. Indirect evidence for flipping of the target adenine by T4Dam has been obtained by fluorescence analysis with substrates containing the base analog 2-aminopurine37. Encouraged by the similar conformations of the Asp-Pro-Pro-Tyr motif in all N6-adenine MTase structures, we superimposed the target adenine of M.TaqI onto T4Dam (Fig. 2e). This superimposition placed the flipped-out adenine with the target amino nitrogen atom occupying the position of a water molecule in the active site. It has been suggested25 that the spinning of this amino group is impeded and its pKa altered by the formation of a pair of hydrogen bonds, one of which is to a MTase backbone carbonyl oxygen. In the case of T4Dam, this would be between Pro172 and Pro173 in the conserved Asp-Pro-Pro-Tyr of motif IV. This particular carbonyl is expected to be fairly rigid as it is between two prolines. The second proton of the target amino group makes a hydrogen bond with the side chain of Asp171. Together these hydrogen bonds may increase the nitrogen electron density and facilitate a nucleophilic attack on the AdoMet methylsulfonium group. In addition, the hydroxyl group of Tyr181 is between the target amino group and the AdoHcy sulfur (Fig. 2e), where the transferable methyl group will be attached in the AdoMet, potentially also facilitating the reaction.
Dam-DNA nonspecific interactions
We were also able to crystallize T4Dam in a ternary complex with AdoHcy and a GATC-containing synthetic DNA duplex. The oligonucleotide, a self-complementary 12-bp palindrome, had the sequence 5′-ACAGGATCCTGT-3′, where the target GATC sequence is centrally located. In the crystal, the DNA duplexes (primarily in B-form) were stacked head-to-end along the crystallographic a axis with an ~15° twist at the joint, forming a pseudo-continuous DNA duplex (Fig. 3a). The identity of the DNA sequence was confirmed by replacing the 5′-end cytosine with 5-iodocytosine. Unexpectedly, a nonspecific loose binding mode38 was observed in the crystal with two Dam monomers (molecules A and B) bound to one DNA duplex (Fig. 3a). Molecule A binds to a single DNA duplex spanning seven base pairs, whereas molecule B binds in the joint of two DNA molecules spanning six base pairs. The buried solvent-accessible surface area39 between protein and DNA is 474 Å2 and 542 Å2 for molecules A and B, respectively. This area is ~3–4× lower than those observed in the specific complexes of M.HhaI–DNA (1,529 Å2) and M.TaqI–DNA (1,886 Å2) and ~2× lower than those observed in the non-sequence-specific protein–DNA complex HMG-D (905 Å2) and for chromosomal protein Sac7d (865 Å2) (ref. 38). The ratio of polar to nonpolar contacts of the buried surface, calculated by using the CONTACT program in CCP4 to determine the contacting atoms and then adding up the polar and nonpolar ones, is ~0.8 for molecule A and 0.6 for molecule B. These ratios are within the range observed for non-sequence-specific DNA recognition (0.5 for HMG and Sac7d and 1.1 for the nucleosome)38.
The loose ternary structure of T4Dam–DNA–AdoHcy. (a) The 12-bp DNA (one strand in blue and the other in red) is stacked head-to-end forming a pseudo long duplex along the crystal a axis with the length of ~40 Å. Molecule A binds to a single DNA molecule, whereas molecule B binds at the joint of two DNA molecules. (b) T4Dam-phosphate interactions. The same regions (residues 129–134) of molecules A and B are shown relative to a single DNA molecule. (c) Schematic diagram of the protein-phosphate interactions. Residues making interactions are black for molecule A and red for molecule B. The asterisks indicate residues from a neighboring symmetry-related molecule.
The two Dam monomer structures in the nonspecific ternary complex are quite similar to each other and to the binary structure, with an r.m.s. deviation of 0.55 Å over all Cα atoms. There are no substantial conformational changes evident after nonspecific binding in the ternary complex. The only obvious differences involve side chain rotamers of several residues (Asn133, Lys134 and Asn135) involved in phosphate interactions. However, the orientations of the two molecules relative to the DNA helical axis are different: molecule A is rotated by ~20° relative to molecule B, resulting in a larger cavity40 for molecule A (170 Å3) vs. molecule B (30 Å3) at the interface of protein and DNA. The smaller cavity and the larger buried surface area suggest that the binding mode of molecule B might be closer to a specific binding mode than that if molecule A.
Protein-phosphate interactions are primarily mediated via the TRD, that is, a stretch of ~20 amino acids from the β-hairpin to the N-terminal end of helix αB5 (Fig. 3b). Both Dam monomers approach the DNA from the minor groove side. In molecule A, Arg130, Asn133, Lys134 and Asn135 interact with the three phosphates (T10, G11 and T12) near the 3′ end of one strand and Lys129 and Lys81 of helix αB3 interact with the two phosphates neighboring the C5 of the target GATC in the complementary strand (Fig. 3c). Molecule B interacts symmetrically with the joint between two DNA molecules by contacting two phosphates flanking the A3 of one DNA molecule and the two phosphates flanking the A10 from the complementary strand of the next DNA duplex (Fig. 3c). Although electrostatic interactions are dominant, Phe111 of the β-hairpin makes van der Waals contacts with the phosphate backbone carbons (C4′ and C5′) of A10. The interactions occur primarily in the minor groove side, the protein structure is maintained and virtually no sequence-specific hydrogen bonds are formed—all features that are also shared in other examples of non-sequence-specific DNA binding38. In addition, the locations of the DNA-interacting residues are in agreement with the solution studies for probing the DNA interface in M.EcoRV29, where DNA interaction residues (specific and nonspecific) have been mapped to the corresponding N-terminal loop, the hairpin and helix αD1.
DISCUSSION
We do not understand why T4Dam failed to bind specifically with GATC sites (which are separated in the crystal by 12 base pairs from center to center). One explanation for the loose binding between T4Dam and DNA in the ternary structure derives from the fact that T4Dam, like E. coli Dam41, methylates DNA with multiple GATC sites in a processive manner; in other words, more than one methyl group may be transferred per bound Dam monomer42. Thus, under single-turnover conditions (Dam:DNA ratio > 1.0), a catalytically competent complex was formed that contained two Dam monomers per synthetic duplex43. Since the rate-limiting step in the overall T4Dam methylation process is the release of product AdoHcy from the enzyme44, it follows that after methyl transfer the Dam–AdoHcy complex must release from the target site, diffuse along the DNA, and AdoMet must exchange for AdoHcy before the next methyl transfer can occur. We suggest that in the ternary crystal structure, the T4Dam–AdoHcy complex may be positioned on the duplex in a fashion that corresponds to the stage following methyl transfer. That is, it is not in contact with the GATC target site; rather it contacts the phosphodiester backbone and is primed for diffusion and/or exchange of AdoHcy with AdoMet. The cofactor cover (the 310-helix αD1 and the flanking loops) is not involved in the nonspecific phosphate backbone interactions. Therefore, it is possible that the cover will be opened up (as observed in the M.DpnII–AdoMet structure) during the cofactor exchange while the enzyme stays bound to the DNA. Notably, restriction-associated MTases are distributive41, because processive methylation of DNA would interfere with the biological function of restriction-modification systems. However, the structural basis for T4Dam processivity is not obvious in comparison with M.DpnII; these two proteins have quite similar structures but differ substantially in their processivity. Breyer and Matthews45 suggested that processive enzymes tend to enclose their substrates to a greater extent. More structural work will be required to settle this point.
METHODS
Protein purification and crystallization
T4Dam was overexpressed and purified to near homogeneity essentially as described previously46. Bacterial cells were lysed and the cell debris was removed by centrifugation. The resulting supernatant (in 20 mM potassium phosphate, pH 7.4, 1 mM EDTA, 0.05% (v/v) β-mercaptoethanol, 400 mM NaCl) was loaded onto a 5-ml HiTrap Q column (Amersham Pharmacia Biotech) and the flowthrough was collected and then loaded onto a 5-ml HiTrap SP column. The protein was eluted from the SP column at ~420–470 mM NaCl. The peak activity fractions were pooled, concentrated and loaded onto a sizing column (equilibrated with 20 mM HEPES, pH 7.4, 1 mM EDTA, 0.05% (v/v) β-mercaptoethanol, 400 mM NaCl); T4Dam migrated as a ~30-kDa globular protein in Superdex S75.
For preparation of the Dam–AdoHcy binary complex, AdoHcy was added to the protein to a final concentration of 0.2 mM and the mixture was incubated on ice for 2 h before it was loaded onto the sizing column. The resulting Dam–AdoHcy complex was concentrated to ~26 mg ml−1 and used for crystallization. Crystals were grown by vapor diffusion at 16 °C as hanging drops using reservoir solution of 2.4 M ammonium sulfate and 100 mM MES pH 6.0.
For preparation of the ternary complex, the Dam–AdoHcy solution was mixed at an ~1:1 molar ratio with annealed DNA oligonucleotide. The crystals with a 12-mer DNA with blunt ends, formed with a reservoir solution of 20–25% (w/v) PEG 8000, 100 mM HEPES, pH 7.5, and 10 mM ammonium sulfate.
Structure determination
We encountered several problems during the course of structural determination of the ternary complex. First, the crystals suffered severe radiation damage, often resulting in incomplete data sets. Second, the cell dimensions changed from crystal to crystal, with variation of up to ~8 Å in the crystallographic b axis (Table 1), making crystals nonisomorphous and different data sets impossible to merge. After screening >100 crystals, we used the following data to solve the structure: two-wavelength anomalous diffraction data at Hg absorption edge (Hg-soaked crystal I) and a single-wavelength (at 1.0 Å) data set from the Hg-soaked crystal II and from crystal III with iodinated oligonucleotide (Table 1).
Table 1
Crystallographic data and refinement statistics
| T4Dam–AdoHcy–DNA
| T4Dam–AdoHcy
| ||||
|---|---|---|---|---|---|
| Crystal I Mercury (Hg) | Crystal II Hg | Crystal III Iodine | Native | ||
| Data Collection | |||||
| Wavelength (Å) | 1.0107 | 0.971 | 1.1 | 1.1 | 1.1 |
| Energy (eV) | 12,266 | 12,768 | 11,271 | 11,271 | 11,271 |
| Space group | P21 | P21 | P21 | P21 | P212121 |
| Unit cell dimensions | |||||
| a (Å) | 40.1 | 40.2 | 39.8 | 39.7 | 58.6 |
| b (Å) | 116.2 | 116.4 | 108.5 | 109.7 | 63.1 |
| c (Å) | 74.9 | 75.0 | 73.4 | 73.6 | 72.7 |
| β (°) | 106.9 | 107.0 | 103.9 | 104.5 | – |
| Resolution range (Å) | 20.0–3.5 | 20.0–3.5 | 20–3.2 | 20–3.1 | 20–2.3 |
| Completeness (%) | 94.0 | 92.3 | 79.7 | 98.6 | 97.6 |
| Rmerge | 7.7 | 8.5 | 11.1 | 9.6 | 5.6 |
| <I/σ> | 10.3 | 8.4 | 7.5 | 13.9 | 16.5 |
| Observed reflections | 20,689 | 19,529 | 20,713 | 43,112 | 56,092 |
| Unique reflections | 8,015 | 8,198 | 8,123 | 11,038 | 12,701 |
| Anomalous sites | 3 | 3 | 3 | 2 | – |
| Highest resolution shell | |||||
| Resolution range (Å) | 3.62–3.5 | 3.63–3.5 | 3.31–3.2 | 3.21–3.1 | 2.35–2.37 |
| Completeness (%) | 89.3 | 90.6 | 75.0 | 97.3 | 99.6 |
| Rmerge | 0.219 | 0.256 | 0.227 | 0.152 | 0.229 |
| <I/σ> | 3.1 | 2.6 | 3.8 | 8.6 | 3.9 |
| Refinement | |||||
| Number of atoms | |||||
| Protein | 1,991 × 2 | 1,991 | |||
| DNA | 486 | – | |||
| AdoHcy | 26 × 2 | 26 | |||
| Water | – | 128 | |||
| Rfactor | 0.238 | 0.222 | |||
| Rfree (5% of the data) | 0.293 | 0.266 | |||
| R.m.s. deviation from ideal | |||||
| Bond lengths (Å) | 0.008 | 0.007 | |||
| Bond angles (°) | 1.4 | 1.3 | |||
| Dihedrals (°) | 23.1 | 22.9 | |||
| Improper (°) | 1.2 | 0.81 | |||
Rmerge = Σ| I − <I>|/ΣI where I is the observed intensity and <I> is the average intensity of multiple symmetry-related observations of the reflection. Rfactor = Σ|Fo − Fc|/Σ|Fo|.
The initial phases were obtained from analysis of the two-wavelength Hg-MAD data of crystal I. SOLVE47 determined three mercury sites whose positions were confirmed by visual examination of the anomalous-difference Patterson map. The phases were improved by maximum-likelihood density modification using RESOLVE48, showing a clear solvent boundary. In parallel, the phases calculated by PHASES49 using the three identical mercury sites were improved by carrying out many rounds of solvent flipping using the program SOLOMON50, which enabled us to identify two protein molecules within an asymmetrical unit bound to a single DNA duplex. The phases were greatly improved by noncrystallographic symmetry averaging along with histogram matching implemented in the program DM51. A partial model was then built and refined by simulated annealing implemented by CNS52. The resulting model was then used to search the molecular replacement solution and the corresponding three mercury sites in crystal II (which provided the cross-averaging matrix between the two crystals) using the program AMoRe53. Using these three mercury sites, a SAD phased map was calculated using SOLVE47. The cross-crystal averaging between MAD (crystal I) and SAD (crystal II) was accomplished using the program DMMULTI51; phases were dramatically improved and a nearly complete model could be built into the density.
The model was refined against the data from crystal III, which contains a 5-iodocytosine at position 2 from the 5′ end. The single electron density peak (6σ above noise level) provided unambiguous determination of the DNA sequence. Numerous cycles of refining using CNS52 and manual rebuilding were conducted. A series of simulated annealing omit maps were used to correct the model. The noncrystallographic restraints were imposed on the two protein molecules throughout the refinement and were only released from protein side chains at the last cycle to account for a different interaction environment between the interfaces of DNA with each molecule.
The structure of the binary complex was readily solved by molecular replacement, using the protein coordinates from the ternary structure. The crystal contains one binary complex in the asymmetric unit.
Solvent-accessible surface areas were calculated using AREAIMOL39 of CCP4 (ref. 54). Contacts of buried surface were calculated using CONTACT54 Cavity sizes were calculated using VOIDOO of RAVE40.
Coordinates
The coordinates of the binary and ternary structures of T4Dam have been deposited in the Protein Data Bank (accession codes 1Q0S and 1Q0T, respectively).
Acknowledgments
We thank beamline staff for help with X-ray data collection at beamlines X12C, X25 and X26C in the facilities of the National Synchrotron Light Source, Brookhaven National Laboratory, R.M. Blumenthal (Medical College of Ohio) for comments and M. Churchill (University of Colorado) for discussion. These studies were supported in part by US Public Health Services grants to S.H and X.C. and the Georgia Research Alliance.
Footnotes
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.