Crystal structures of a template-independent DNA polymerase: murine terminal deoxynucleotidyltransferase

Abstract

The crystal structure of the catalytic core of murine terminal deoxynucleotidyltransferase (TdT) at 2.35 A resolution reveals a typical DNA polymerase beta-like fold locked in a closed form. In addition, the structures of two different binary complexes, one with an oligonucleotide primer and the other with an incoming ddATP-Co(2+) complex, show that the substrates and the two divalent ions in the catalytic site are positioned in TdT in a manner similar to that described for the human DNA polymerase beta ternary complex, suggesting a common two metal ions mechanism of nucleotidyl transfer in these two proteins. The inability of TdT to accommodate a template strand can be explained by steric hindrance at the catalytic site caused by a long lariat-like loop, which is absent in DNA polymerase beta. However, displacement of this discriminating loop would be sufficient to unmask a number of evolutionarily conserved residues, which could then interact with a template DNA strand. The present structure can be used to model the recently discovered human polymerase mu, with which it shares 43% sequence identity.

Fig. 1. General structure and domain organization of TdT. (A) Linear arrangement of the different domains of C-TdT (blue), compared with pol β. The two motifs C and A and the two HhH consensus sequences are indicated in red and green, respectively. The disordered part of C-TdT in the crystal is in cyan and the N-terminal extension of C-TdT compared with pol β is in magenta. The part of the molecule absent from the crystallized construct is in white. (B) General architecture of the TdT structure drawn with Molscript (Kraulis, 1991). The three catalytic aspartate residues are shown in ball-and-stick representation. The N- and C-termini are in dark blue, while the two strictly conserved stretches of sequences implicated in the binding of the incoming dNTP are in cyan and magenta. Loop1 is shown in yellow. An intrinsic magnesium ion (grey) as well as the two putative sodium ions bound to the HhH motifs are represented in CPK mode (blue); only the Na⁺ ion with ligands in an octahedral geometry is labelled.

Fig. 1. General structure and domain organization of TdT. (A) Linear arrangement of the different domains of C-TdT (blue), compared with pol β. The two motifs C and A and the two HhH consensus sequences are indicated in red and green, respectively. The disordered part of C-TdT in the crystal is in cyan and the N-terminal extension of C-TdT compared with pol β is in magenta. The part of the molecule absent from the crystallized construct is in white. (B) General architecture of the TdT structure drawn with Molscript (Kraulis, 1991). The three catalytic aspartate residues are shown in ball-and-stick representation. The N- and C-termini are in dark blue, while the two strictly conserved stretches of sequences implicated in the binding of the incoming dNTP are in cyan and magenta. Loop1 is shown in yellow. An intrinsic magnesium ion (grey) as well as the two putative sodium ions bound to the HhH motifs are represented in CPK mode (blue); only the Na⁺ ion with ligands in an octahedral geometry is labelled.

Fig. 2. Multialignment of TdT and pol µ sequences. In addition to the murine TdT (TdT_mouse) studied here, the multialignment includes the bovine (TdT_bovin), human (TdT_human), chicken (TdT_chick), Xenopus (TdT_xenla), axolotl (TdT_axolo), opossum (TdT_oposs) and trout (TdT_trout) TdT enzymes as well as the human (polm_hsap) and mouse (polm_mmus) polymerase µ. The sequences were divided into two groups: the eight TdT sequences and the two pol µ sequences. Those residues >80% conserved in each group but not in all sequences are coloured in yellow. Strictly conserved residues in each group are red; they are boxed (red) when conserved in all sequences. The sequence of human pol β (polb_hsap) is displayed but not taken into account for calculating conservation. The multiple alignment was obtained with the program Pileup (University of Wisconsin, 1997) using an opening and extension gap penalty of 8 and 2, respectively, and then displayed using the program ESPript (Gouet et al., 1999). The numbering corresponds to the murine TdT sequence. The secondary structure of C-TdT is displayed on top of the alignment as well as that of the aligned human pol β sequence (polb_hsap). Important structural features are indicated at the bottom of the alignment: the N- and C-terminal regions that are close in space are indicated by a green full circle; the three catalytic aspartate residues of motifs C and A are highlighted by full red circles, the two HhH regions by green upper triangles, and loop1 by black circles. R336, G452 and S453 are underlined, with blue upright triangles to indicate that the side chain of R336 binds to the carbonyl oxygen of G452 and helps to maintain the cis-peptide bond between G452 and S453; the two highly conserved regions to which these last three residues belong, forming together the binding site of the incoming dNTP, are singled out with blue full circles. Cysteines are indicated by a blue star. Finally, the location of the insertion present in the longer form of murine TdT is indicated by two red upright triangles.

Fig. 3. C-TdT is in a closed form. Stereo view of the superimposed C_α traces of TdT (red) with the closed form of pol β (blue).

Fig. 4. The binary complex with the incoming nucleotide. (A) Electron density for the binary complex obtained by soaking TdT native crystals with CoCl₂ (10 mM) and ddATP (1 mM). The anomalous difference map at 4.5 Å resolution contoured at 4.5σ is shown in cyan along with the two cobalt atoms (CPK spheres, cyan). The three residues closest to the base (Trp450, Lys403 and Asn474) are shown as ball-and-stick representations. (B) F_obs – F_calc omit map of the final model in the incoming nucleotide-binding site region, contoured at 2.5σ. The three catalytic aspartate residues of motifs C and A as well as Arg432, which forms an ion pair with one of them, Asp434, are represented in ball-and-stick. (C) Comparison of the binding mode of the incoming nucleotide in pol β (blue) and TdT (red): the electron density of the omit map (green) displayed in (B) is superimposed on the ball-and-stick model of the incoming ddCTP (black) of the ternary complex of pol β (Sawaya et al., 1997). (D) Detailed view of the active site with the incoming ddATP in the context of the local secondary structure. The three conserved aspartates of motif C and motif A are shown, as well as the two cobalt ions (CPK spheres, cyan). The highly conserved regions among TdT sequences are coloured purple and green for the helices (α11 and α13) and cyan for the loops. Loop1 is in yellow. The C-terminus is in dark blue.

Fig. 5. The binary complex with the primer strand. (A) Isomorphous density map at 3.0 Å resolution contoured at 4.0σ (magenta), showing density for the three phosphates of sites P1, P2 and P3. The final oligonucleotide model occupying these three sites is drawn as a ball-and-stick representation. The anomalous electron density coming from the anomalous signal of the bromine atoms is in green (4.0σ). (B) F_obs – F_calc omit electron density map in the primer-binding site region (2.5σ). The last base in the Incoming binding site is not stacked with the preceding base. (C) Comparison of the binding mode of the primer strand in pol β (blue) and in TdT (red): the isomorphous Fourier difference map of the TdT binary complex (magenta) is superimposed to the primer strand of the ternary complex of pol β (black) (Sawaya et al., 1997), showing that the phosphates are located in almost the same place in the two proteins. (D) Detailed view of the stabilization of the primer phosphate at site P2, through the Na⁺ ion (yellow CPK sphere) located at the corner of the HhH motif, exactly as in the closed pol β structure (Pelletier et al., 1996). The electron density of the phosphate, calculated as in (a), is in magenta. Drawn with Bobscript (Esnouf, 1999).

Fig. 6. Incoming nucleotide and primer strand in the context of the molecular surface of C-TdT (drawn with Grasp; Nicholls et al., 1991). Exposed aspartate and glutamate residues are in red, while exposed arginine and lysine residue are in blue. (A) View down the oligonucleotide axis, with the incoming nucleotide in place. (B) View from the side with the final oligonucleotide model. The cavity through which the dNTP diffuses to reach the catalytic site is visible at the back.

Fig. 7. Loop1. (A) Stabilization of loop1 through a central water molecule connecting Asp473 of the β–turn–β region of the C-terminal domain with Ser392 of loop1. The direct interaction of the side chain of His475 of the DNH motif with the carbonyl atom of Lys394 of loop1 is also shown. (B) Comparison of C-TdT and pol β in the vicinity of loop1. Loop1 is shown as a C_α trace (magenta) with its characteristic lariat-like structure. The underlying β-sheet of the C-terminal domain is also shown (magenta), superimposed with the same domain of the closed form of pol β (green). The five-residue insertion of pol β compared with TdT observed in this region is thus made visible. There is a deletion in TdT just before helix α12, as compared with pol β, making room for an alternative conformation of loop1 (cyan). (C) Molecular surface of TdT drawn with Grasp (Nicholls et al., 1991) together with the putative model of a primer–template duplex, derived from the human pol β ternary complex (Sawaya et al., 1997). The primer strand is coloured according to the chemical nature of its atoms, while the template strand is in dark blue. The exposed surface due to residues from loop1 is in magenta. It is clear from this view that the duplex part of the template strand cannot be accommodated by TdT.

Fig. 7. Loop1. (A) Stabilization of loop1 through a central water molecule connecting Asp473 of the β–turn–β region of the C-terminal domain with Ser392 of loop1. The direct interaction of the side chain of His475 of the DNH motif with the carbonyl atom of Lys394 of loop1 is also shown. (B) Comparison of C-TdT and pol β in the vicinity of loop1. Loop1 is shown as a C_α trace (magenta) with its characteristic lariat-like structure. The underlying β-sheet of the C-terminal domain is also shown (magenta), superimposed with the same domain of the closed form of pol β (green). The five-residue insertion of pol β compared with TdT observed in this region is thus made visible. There is a deletion in TdT just before helix α12, as compared with pol β, making room for an alternative conformation of loop1 (cyan). (C) Molecular surface of TdT drawn with Grasp (Nicholls et al., 1991) together with the putative model of a primer–template duplex, derived from the human pol β ternary complex (Sawaya et al., 1997). The primer strand is coloured according to the chemical nature of its atoms, while the template strand is in dark blue. The exposed surface due to residues from loop1 is in magenta. It is clear from this view that the duplex part of the template strand cannot be accommodated by TdT.

Fig. 7. Loop1. (A) Stabilization of loop1 through a central water molecule connecting Asp473 of the β–turn–β region of the C-terminal domain with Ser392 of loop1. The direct interaction of the side chain of His475 of the DNH motif with the carbonyl atom of Lys394 of loop1 is also shown. (B) Comparison of C-TdT and pol β in the vicinity of loop1. Loop1 is shown as a C_α trace (magenta) with its characteristic lariat-like structure. The underlying β-sheet of the C-terminal domain is also shown (magenta), superimposed with the same domain of the closed form of pol β (green). The five-residue insertion of pol β compared with TdT observed in this region is thus made visible. There is a deletion in TdT just before helix α12, as compared with pol β, making room for an alternative conformation of loop1 (cyan). (C) Molecular surface of TdT drawn with Grasp (Nicholls et al., 1991) together with the putative model of a primer–template duplex, derived from the human pol β ternary complex (Sawaya et al., 1997). The primer strand is coloured according to the chemical nature of its atoms, while the template strand is in dark blue. The exposed surface due to residues from loop1 is in magenta. It is clear from this view that the duplex part of the template strand cannot be accommodated by TdT.