Structure of D-63 from sulfolobus spindle-shaped virus 1: surface properties of the dimeric four-helix bundle suggest an adaptor protein function

Abstract

Sulfolobus spindle-shaped virus 1 (SSV1) and its fusellovirus homologues can be found in many acidic (pH <or= 4.0) hot springs (>or=70 degrees C) around the world. SSV1 contains a 15.5-kb double-stranded DNA genome that encodes 34 proteins with greater than 50 amino acids. A site-specific integrase and a DnaA-like protein have been previously identified by sequence homology, and three structural proteins have been isolated from purified virus and identified by N-terminal sequencing (VP1, VP2, and VP3). The functions of the remaining 29 proteins are currently unknown. To assign functions to these proteins, we have initiated biochemical and structural studies on the SSV1 proteome. Here we report the structure of SSV1 D-63. The structure reveals a helix-turn-helix motif that dimerizes to form an antiparallel four-helix bundle. Mapping residues conserved among three fusellovirus isolates onto the structure shows that one face of the rod-shaped molecule is highly conserved. This conserved surface spans the dimer axis and thus exhibits 2-fold symmetry. Two smaller conserved patches, also related by 2-fold symmetry, are found on the opposite face of the molecule. All of these conserved surfaces are devoid of clefts or pockets typically used to bind small molecules, suggesting that D-63 may function as an adaptor protein in macromolecular assembly.

FIG. 1.

(A) The D-63 homodimer forms an antiparallel four-helix bundle. Monomers A and B are each shown as a ribbon diagram depicting secondary structure elements. Blue, monomer A; amber, monomer B. The 2-fold symmetry axis (double-headed arrow) runs horizontally between the two monomers relating the N and C termini of the blue monomer at the bottom to the N and C termini of the amber monomer at the top. (B) The dimer interface is hydrophobic. Monomer A is depicted as a blue worm, while monomer B is shown in amber and gray. All surface residues are amber, with the exception of residues Phe¹¹, Leu¹⁴, Val¹⁸, Leu²¹, Ile²⁵, Ile²⁸, Leu⁴², Ala⁴⁵, Val⁴⁹, Ile⁵², Leu⁵⁶, and Leu⁵⁹. These residues are gray and illustrate the extent of the hydrophobic surface found at the dimer interface.

FIG. 2.

(A) Sequence alignments of D-63 fusellovirus homologues. Strictly conserved residues are red, and nonconserved residues are black. Secondary structure assignments, i.e., the extent of the alpha helices, are indicated above the sequence. The hydrophobic residues occur within the alpha helices in a clear heptad repeat, (ABCDEFG)_n, with hydrophobic residues at positions A and D. SSV2 was isolated from Sulfolobus solfataricus strain REY15/4 from Iceland (23). SSV RH was isolated from Yellowstone National Park (26). (B) Conserved surface residues among D-63 homologues. Conserved basic, acidic, polar, and nonpolar residues are blue, red, cyan, and gray, respectively. Nonconserved residues are yellow. Strong conservation of SSV1, SSV2, and SSV RH surface residues is seen for the N-terminal face of the four-helix bundle. The twofold symmetry axis runs perpendicular to this face through the center of the four-helix bundle. Thus the conserved surface exhibits 2-fold symmetry. Conserved residues contributing to this surface are labeled for one of the two monomers. The second monomer is related to the first by the 2-fold symmetry axis. This putative ligand binding surface is further enlarged when conservative substitutions are also mapped to the surface (not shown). The surface is a mixture of both polar and nonpolar residues.