Visualizing ATP-dependent RNA translocation by the NS3 helicase from HCV - PubMed (original) (raw)

Visualizing ATP-dependent RNA translocation by the NS3 helicase from HCV

Todd C Appleby et al. J Mol Biol. 2011.

Abstract

The structural mechanism by which nonstructural protein 3 (NS3) from the hepatitis C virus (HCV) translocates along RNA is currently unknown. HCV NS3 is an ATP-dependent motor protein essential for viral replication and a member of the superfamily 2 helicases. Crystallographic analysis using a labeled RNA oligonucleotide allowed us to unambiguously track the positional changes of RNA bound to full-length HCV NS3 during two discrete steps of the ATP hydrolytic cycle. The crystal structures of HCV NS3, NS3 bound to bromine-labeled RNA, and a tertiary complex of NS3 bound to labeled RNA and a non-hydrolyzable ATP analog provide a direct view of how large domain movements resulting from ATP binding and hydrolysis allow the enzyme to translocate along the phosphodiester backbone. While directional translocation of HCV NS3 by a single base pair per ATP hydrolyzed is observed, the 3' end of the RNA does not shift register with respect to a conserved tryptophan residue, supporting a "spring-loading" mechanism that leads to larger steps by the enzyme as it moves along a nucleic acid substrate.

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Figures

Fig. 1

Fig. 1

Structure of Full-length HCV NS3 bound to RNA. (a) Cartoon representation of NS3 viewed from the side. NS3hel domains 1 (D1), domain 2 (D2), and domain 3 (D3) are colored yellow, green, and orange, respectively. NS3pro (gray) with N-terminally-fused NS4A peptide (red) lies beneath NS3hel in this orientation. N and C termini and ssRNA are labeled accordingly. (b) A top view of NS3 (colored as above). NS3pro is removed for clarity. The bound sulfate ion is shown as a space-filling model, while the 5’ and 3’-ends of the ssRNA (stick model) are labeled accordingly. (c) Conserved SF2 helicase motifs mapped onto NS3. Motif I (residues 204–211), motif Ia (residues 230–235), motif Ib (residues 268–272), motif II (residues 290–293), motif III (residues 322–324), motif IV (residues 365–372), motif V (residues 411–419), and motif VI (460–467) are colored according to the legend. Tyr 241 (motif Y) is shown as a stick model.

Fig. 2

Fig. 2

Electron density maps for selected ligands. Regions of the 2Fo-Fc maps (light blue) are shown for bound RNA in (a) the NS3-rU8Br4 structure (2.0Å) and (b) the NS3rU8Br4/ADP•BeF3 structure (2.3Å). Fo-Fc density calculated prior to adding bromine to U4 nucleotide residues in both models is contoured at 4σ and shown in red. (c) A 2Fo-Fc density map (light blue) for ADP•BeF3 bound to NS3 (2.05Å) is superimposed on the final model of the nucleotide, magnesium ion, and two coordinating water molecules.

Fig. 3

Fig. 3

HCV NS3 binding to single-stranded nucleic acid. (a) Close-up view of ssRNA bound to NS3. RNA is shown as sticks colored by atom type (carbon, cyan; nitrogen, dark blue; oxygen, red; phosphate, orange; bromine, burgundy). NS3 is shown as a transparent surface and colored by domain (as in Fig. 1). The side chain or backbone atoms of NS3 residues interacting with the RNA are represented by sticks and are labeled accordingly. The side chains of Lys 371 and Arg 393 are disordered in the structure and omitted here for clarity. Nucleotide residues (U3–U7) and the 5’ and 3’-ends of the strand are labeled. (b) Overlay of the NS3-rU8Br4 and NS3hel-ssDNA (

PDB ID: 3KQH

) structures. RNA is shown as above, while the DNA is shown with carbons colored yellow. Protein residues in motif V of NS3-rU8Br4 (cyan) and NS3hel-ssDNA (yellow) are shown as cartoon coils. Leu 414 of NS3hel-ssDNA and the side chain atoms for Thr 416 and Trp 501 in both structures are represented by sticks. Hydrogen bonds between protein atoms and the phosphodiester backbone linking nucleotides 4 and 5 are represented by red dashed lines. Distances between corresponding phosphoryl oxygens of DNA and RNA are indicated by dashed lines (black).

Fig. 4

Fig. 4

(a) Nucleotide induced structural rearrangement of NS3. The structure of nucleotide-free NS3-rU8Br4 (left) compared to NS3-rU8Br4/ADP•BeF3 (right). NS3 represented by cartoon ribbons with transparent surfaces while RNA is shown as sticks. Upon binding to ADP•BeF3 (shown as spheres), D1 (yellow) and D2 (green) pivot towards each other, collapsing around the nucleotide. Arrows indicate the distance between the Cα carbons of residues Lys 244 (D1) and Gly 468 (D2) before and after ADP•BeF3 binding. (b) Close-up of the NS3 ATPase site. Specific interactions between ADP•BeF3 and the two NS3 domains, D1 (right panel) and D2 (left panel), are diagramed separately for clarity. D1 residues (yellow carbons) in direct contact with either ADP•BeF3 or the Mg2+ ion are shown as sticks and labeled accordingly. D2 residues (green carbons) interacting with the nucleotide analogue are also labeled. Gln 460 from D2, which coordinates a water molecule near the γ-position of the nucleotide, is also shown.

Fig. 5

Fig. 5

Nucleotide induced conformational changes in NS3 lead to differences in RNA binding. (a) Structural overlay of nucleotide-free NS3-rU8Br4 (colored according to Fig. 1) and NS3-rU8Br4/ADP•BeF3 (gray). (b) Local conformational change in of motif V of D2. Motif V (residues 411–419) in the nucleotide-free structure (green helical coil and sticks) rearranges upon binding to ADP•BeF3 (spheres), resulting in the repositioning of Thr 416. For clarity, only U5 of the RNA is shown (cyan sticks, nucleotide-free structure; gray sticks, nucleotide-bound structure). (c) Comparison of RNA binding to NS3 in the nucleotide-bound and nucleotide-free states. The nucleotide-bound structure (+Nuc, gray coil and sticks) and the nucleotide-free structure (−Nuc, green coils and cyan sticks) are overlaid. Thr 416, Trp 501 and RNA nucleotides are labeled accordingly.

Fig. 6

Fig. 6

Schematic representation of nucleotide induced changes in RNA binding during a single round of ATP hydrolysis. The colored boxes represent D1 (yellow), D2 (green), and D3 (orange). Specific NS3 residues and atoms involved in binding RNA are labeled and shown to interact with either the uridine bases (light orange hexagons) or the phosphodiester backbone (red circles) of the RNA strand. Ribose sugars (gray pentagons) are numbered from 5’→3’ based on the position of the bromine-labeled U4 in both structures (labeled with small purple hexagons). The straight arrow indicates the relative movement of NS3 with respect to RNA.

Fig. 7

Fig. 7

Schematic representation of NS3 stepping along an RNA tracking strand during multiple rounds of the ATPase cycle. Domains 1, 2, and 3 are colored yellow, green and orange respectively. The phosphodiester backbone of the tracking strand is shown as small circles labeled by nucleotide residue number (P7-P2, 5’→3’). The small, upside down triangles represent conserved Thr 269 of D1 (yellow) and Thr 411 of D2 (green). Three rounds of ATP binding and release are depicted (labeled 1–3).

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