Structure of a baculovirus sulfhydryl oxidase, a highly divergent member of the erv flavoenzyme family

Abstract

Genomes of nucleocytoplasmic large DNA viruses (NCLDVs) encode enzymes that catalyze the formation of disulfide bonds between cysteine amino acid residues in proteins, a function essential for the proper assembly and propagation of NCLDV virions. Recently, a catalyst of disulfide formation was identified in baculoviruses, a group of large double-stranded DNA viruses considered phylogenetically distinct from NCLDVs. The NCLDV and baculovirus disulfide catalysts are flavin adenine dinucleotide (FAD)-binding sulfhydryl oxidases related to the cellular Erv enzyme family, but the baculovirus enzyme, the product of the Ac92 gene in Autographa californica multiple nucleopolyhedrovirus (AcMNPV), is highly divergent at the amino acid sequence level. The crystal structure of the Ac92 protein presented here shows a configuration of the active-site cysteine residues and bound cofactor similar to that observed in other Erv sulfhydryl oxidases. However, Ac92 has a complex quaternary structural arrangement not previously seen in cellular or viral enzymes of this family. This novel assembly comprises a dimer of pseudodimers with a striking 40-degree kink in the interface helix between subunits. The diversification of the Erv sulfhydryl oxidase enzymes in large double-stranded DNA viruses exemplifies the extreme degree to which these viruses can push the boundaries of protein family folds.

Fig. 1.

Ac92 exhibits a new quaternary structure for Erv enzymes. The FAD is shown as orange sticks, and cysteine side chains are balls and sticks. The CXXC label indicates the position of the active-site dicysteine motif adjacent to the FAD. (A) Each polypeptide chain is colored from red (amino terminus; N) to blue (carboxy terminus; C). Ac92 dimerizes via the carboxy terminus of its two helical bundle domains. The three views of the dimer are related by successive rotations around the horizontal axis. (B) Both domains of Ac92 have the helical bundle topology of the Erv family enzymes. The two helical bundles of the Ac92 protomer (Ac92-N and Ac92-C are the amino- and carboxy-terminal bundles, respectively) are compared to the structure of a subunit of a cellular Erv family sulfhydryl oxidase, the S. cerevisiae Erv2 protein (ScErv2p; PDB code 1JR8). The four primary helices in each domain are colored red, yellow, green, and blue in order from amino to carboxy terminus. Two views of each domain, related by rotation around the horizontal axis, are shown. (C) The Ac92 dimer interface has a unique geometry. The 2-fold axis of the dimer runs through the black symbol in the center of each panel. In the left panel, one subunit is light gray and the second is dark gray, with the baculovirus-specific insertions in pink and red. Within these insertions, the secondary structural elements of the β-α-β motif are labeled (underlined symbols), and a spiral symbol is placed within the loop enclosed by this motif. The view in the right panel was obtained by rotating 180° around the vertical axis and zooming in. The red and pink arrows indicate the amino- to carboxy-terminal directionality of the helices, and the longer arrows emphasize the collision course of the α3 helices prior to the kink. Putative hydrogen bonds are shown as red dashed lines, and a potential cation-π interaction stabilizing the geometry in this region is represented with a blue cone labeled cat π.

Fig. 2.

Analytical equilibrium ultracentrifugation shows Ac92 to be a dimer in solution. The measured solution molecular mass was 59 kDa; it was calculated to be 63 kDa based on amino acid sequence. In the bottom panel, two representative 280-nm absorbance traces, taken at 17,000 and 23,000 rpm from a sample at approximately 10 μM protomer concentration, are shown as open circles. The results of the global fit to the entire data set (see Materials and Methods) are shown as black curves. The upper two panels show the residuals between the data and the fit for each of these two traces. The inset in the lower panel shows that the slope of the plot of the natural logarithm of the absorbance versus the square of the radius corresponds to that calculated for a dimer. Calculated slopes for monomeric (1), dimeric (2), trimeric (3), and tetrameric (4) species are indicated.

Fig. 3.

Pseudosymmetry in Ac92 suggests an evolutionary pathway for the formation of its novel quaternary structure. (A) One subunit of the Ac92 dimer is compared to a homodimer of S. cerevisiae Erv2p (ScErv2p). The redox-active domain of Ac92, in various colors, packs against the redox-inactive domain, in beige, in a manner similar to how the two subunits of dimeric cellular Erv enzymes self associate. In the ScErv2p structure, one subunit is shown in various colors and the second in beige. (B) Proposed evolutionary route to the Ac92 complex architecture. A homodimeric precursor may have undergone domain duplication and fusion to generate a single-chain pseudodimer. One of the domains then lost activity (light gray). The mutation of the redox-active domain (dark gray triangle) may then have enabled the dimerization of the pseudodimer.

Fig. 4.

Ac92 active-site and surface properties. (A) The active-site region of Ac92 is shown with the side chain atoms of the CXXC motif in spheres at half their van der Waals radii. The FAD is in orange sticks, and the side chains of residues making key electrostatic interactions (dotted red lines) with the FAD also are shown as sticks. (B) An electrostatic surface representation of the Ac92 dimer is shown with regions of basic potential colored blue (>10 k_BT/e) and acidic regions red (<−10 k_BT/e, where k_B is the Boltzmann constant, T is the absolute temperature, and e is the electron charge). The spring labeled 20 to 25 Å in the top panel represents the variability seen in the distance between the walls of the groove in the Ac92 structure when the P1 and C2 crystal structures were compared. The approximate locations of the Ac92-N and Ac92-C domains and their symmetry-related copies (Ac92-N′ and Ac92-C′) are indicated. In the bottom view, yellow arrows point at the active-site disulfides and approximate the direction from which substrate thiols would approach.

Fig. 5.

Three viral sulfhydryl oxidases use three orthogonal interfaces for dimerization. A topology diagram demonstrating the helices buried in the interface of the FAD-binding, sulfhydryl oxidase domain for each viral enzyme dimer is shown above a representation of the dimer structure, with helices shown as cylinders. The four primary helices in each bundle are shown in red, yellow, green, and blue from amino to carboxy terminus. The ASFV structure is shown looking down the 2-fold axis of symmetry. The mimivirus and baculovirus structures are presented with the 2-fold axis vertical in the plane of the page.