Crystal Structure of the Superfamily 1 Helicase from Tomato Mosaic Virus

Comparison of the ToMV-Hel structure with those of nonviral SF1 helicases. (Left) The three-dimensional structures shown as ribbon diagrams. Domains 1A and 2A, which form the helicase cores, are colored green and blue, respectively. N-terminal domains are shown in yellow. Accessory domains are shown in light orange and orange. (Right) Topology diagrams of the helicase core domains. DNAs are shown in gray. The positions of the 12 conserved motifs defined by Fairman-Williams and colleagues (9) are marked. The sequences for the motifs labeled in gray are absent in the corresponding proteins. α-helices and β-sheets are shown as cylinders and arrows, respectively. The representative structures of the three nonviral SF1 helicases that have the highest Z scores against ToMV-Hel core domain in the DALI (15) conservation list (see Table S3 in the supplemental material) are shown in panels B, C, and D. (A) ToMV-Hel. (B) Upf1 (PDB code 2GK7; Z score, 21.7; root mean square deviation [RMSD], 3.3 Å). (C) UvrD (PDB code 2IS1; Z score, 18.4; RMSD, 3.1 Å). (D) RecD2 (PDB code 3GPL; Z score, 15.2; RMSD, 3.7 Å). The diagrams were created using PyMol (http://pymol.org/pymol).

NTP-binding sites. (A) The putative amino acid residues comprising the NTPase active site in the ToMV-Hel–sulfate complex structure. (B) The AMPPNP interaction site in hUpf1 (PDB code 2GJK) (5). (C) The ADPNP interaction site in PcrA (PDB code 3PJR) (47). The structures are shown as ribbon diagrams. The residues that interact with the sulfates in ToMV-Hel, with AMPPNP in hUpf1, and with ADPNP in PcrA are shown as magenta stick models and are labeled by residue number. AMPPNP and ADPNP are shown as a yellow stick models. The diagrams were created using PyMol.

Putative RNA-binding sites. (A) Electrostatic surface of the ToMV-Hel structure. Positive, negative, and uncharged residues are shown in blue, red, and white, respectively. The putative RNA-binding site is a shallow groove indicated by the black arrow. The N-terminal-domain β-sheet, which is positively charged, is indicated by the red arrow. (B) The positions of the ToMV amino acid residues comprising the putative RNA-binding site are structurally homologous to those found in nonviral SF1 helicase motifs that interact with DNA. (C) Interactions between PcrA and DNA in the PcrA binary complex (PDB code 2PJR). (D) Interactions between RecD2 and DNA in the RecD2 binary complex (PDB code 3GP8) (40). Conserved and nonconserved residues that recognize DNA or RNA are shown as blue and magenta stick models, respectively. DNA is shown as a yellow stick model in panels C and D. The diagrams were created using PyMol.

Locations of previously characterized mutations in tobamovirus helicase domains. The residues that are important for interaction with the N protein (46), with PAP1 (36), and with Tm-1 (31, 44) are shown as green, orange, and magenta stick and surface models and are labeled by residue name and number.

Interactions between the ToMV helicase domain and host proteins. (A) Start and stop positions of the ToMV-Hel deletion mutants used in the split-ubiquitin-based yeast two-hybrid assay. (B) Interactions between TOM1 and ToMV-Hel deletion mutants assessed by the split-ubiquitin-based yeast two-hybrid assay. (C) Interaction between ARL8 and ToMV-Hel deletion mutants assessed by the split-ubiquitin-based yeast two-hybrid assay. (D) Effect of the long α-helix from the ToMV-Hel N-terminal domain on the interaction of TOM1 with ARL8 as assayed by the split-ubiquitin-based yeast three-hybrid system. Averages and standard deviations of β-galactosidase activity (Miller units) for three or four independent yeast transformants are shown in panels B to D.