Insights from the structure of a smallpox virus topoisomerase-DNA transition state mimic - PubMed (original) (raw)

Insights from the structure of a smallpox virus topoisomerase-DNA transition state mimic

Kay Perry et al. Structure. 2010.

Abstract

Poxviruses encode their own type IB topoisomerases (TopIBs), which release superhelical tension generated by replication and transcription of their genomes. To investigate the reaction catalyzed by viral TopIBs, we have determined the structure of a variola virus topoisomerase-DNA complex trapped as a vanadate transition state mimic. The structure reveals how the viral TopIB enzymes are likely to position the DNA duplex for ligation following relaxation of supercoils and identifies the sources of friction observed in single-molecule experiments that argue against free rotation. The structure also identifies a conformational change in the leaving group sugar that must occur prior to cleavage and reveals a mechanism for promoting ligation following relaxation of supercoils that involves an Asp-minor groove interaction. Overall, the new structural data support a common catalytic mechanism for the TopIB superfamily but indicate distinct methods for controlling duplex rotation in the small versus large enzyme subfamilies.

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Figures

Figure 1

Variola virus TopIB-DNA-vandate complex. (A) Overall structure of the complex. NTD=N-terminal domain; CTD=C-terminal domain. (B) Electron density within the active site following refinement. The density is from a σA-weighted 2Fo-Fc map contoured at 1.2σ. (C) Sequence of the DNA duplex used to form the vTopIB-DNA-vanadate complex with the core recognition/activation sequence shaded gray. The vanadate linkage is indicated by ‘v’, with covalent attachment of Tyr274 indicated. The numbering indicated is used throughout the text. In all panels, DNA downstream of the cleavage site is colored yellow and the vanadium linkage is highlighted in brown and red. Figures 1a,b, 2a, 3, 5, & 6 were produced using Pymol (Delano, 2002).

Figure 2

Downstream DNA contacts. (A) View of the DNA downstream of the cleavage site with interactions illustrated for α10a, α9, the amino-terminal domain, and the β7- β8 loop. The α5 and α10a helices are bound in adjacent major grooves flanking the cleavage site. (B) Schematic of vTopIB-DNA contacts downstream of the cleavage site. Dashed lines indicate hydrogen bonds and solid lines indicate van der Waals contacts. The downstream DNA in panels A and B is colored yellow and includes part of an adjacent duplex related by crystallographic symmetry (see Figure S1). (C) Sequence alignment of seven poxvirus TopIBs, one bacterial TopIB, human TopIB, and the two subunits of trypanosomal TopIB in the α8-α10 region. Secondary structure assignments are for variola virus TopIB. Protein sequence accession numbers are Var: variola (NP_042133), Swi: swinepox (NP_570234), Fow: fowlpox (NP_039106), Mol: mollescum contagiosum(NP_044038), Ect: Ectromelia (NP_671606), Myx: Myxoma (NP_051788), Cro: crocodilepox (YP_784296), Dr: Deinococcus radiodurans (NP_294413), Hs: human (NP_003277), LdL (AAF73185)/LdS (ABW86320): large and small subunits of Leishmania donovani, respectively. Conserved TopIB residues are shaded yellow and residues likely to be involved in downstream contacts in the small TopIB enzymes are shaded gray.

Figure 3

Comparison of (A) variola virus TopIB and (B) L. donovani TopIB (PDB code 2B9S) transition state mimic complexes. The view orientations of the two complexes are identical with respect to the conserved active site residues. Protein regions that contact DNA downstream of the cleavage site are colored green. In (A), the DNA is bent by 35-40°, resulting in a trajectory that facilitates interaction with the N-terminus of α9. Note the different orientations of the α9 helix in the two complexes. The downstream DNA in panels A and B is colored yellow and in panel A the downstream DNA includes part of an adjacent duplex related by crystallographic symmetry (see Figure S1).

Figure 4

Active site of the vTopIB-DNA transition state mimic. (A) Stereo diagram showing the five conserved active site residues and three water molecules most closely associated with the reaction center. The three water molecules and Lys167 form a chain linking Tyr274 and O5′ of the -1Ade residue. (B) Schematic of the active site showing all of the hydrogen-bonding interactions made by the residues in (A). Solvent molecules are indicated by red and gray circles. See Fig. S2 for a comparison of active site hydrogen-bonding distances for the non-covalent, covalent, and transition state mimic structures.

Figure 5

Conformation of the -1 sugar during TopIB catalysis. (A) The -1Ade sugar in the vTopIB-DNA-vanadate complex. The backbone torsion angle γ (the C3′-C4′-C5′-O5′ dihedral angle) adopts a trans configuration, placing O5′ in position for axial coordination of vanadium and in position for hydrogen-bonding by Lys167 and Arg130. The sugar also adopts an RNA-like C3′-endo pucker. (B) The corresponding sugar in a pre-cleavage complex of human TopIB (Y723F mutant) bound to DNA (PDB code 1A35). Here, the γ angle is close to the standard value of -60°, and the sugar is C2′-endo. The phosphate has not yet rotated and O5′ is not in position for interaction with Lys532 and Arg488.

Figure 6

A role for Asp168 in strand ligation. Asp168 makes short van der Waals or CH-O hydrogen bonding contacts to the −1 and +2 sugars located on opposite faces of the minor groove. The β7-β8 loop is anchored to the upstream DNA via four distinct interactions involving the backbone and side chain of Asp168 and the side chain of Lys167. Lys167 and Asp168 in turn provide a docking surface for the -1 sugar, where interactions with the ribose ring and O5′ may promote a favorable conformation for efficient ligation. Asp168 contacts several ribose ring atoms with distances < 3.5 Å; the closest contacts are shown.

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Insights from the structure of a smallpox virus topoisomerase-DNA transition state mimic - PubMed (original) (raw)