The structure of the poxvirus A33 protein reveals a dimer of unique C-type lectin-like domains

Abstract

The current vaccine against smallpox is an infectious form of vaccinia virus that has significant side effects. Alternative vaccine approaches using recombinant viral proteins are being developed. A target of subunit vaccine strategies is the poxvirus protein A33, a conserved protein in the Chordopoxvirinae subfamily of Poxviridae that is expressed on the outer viral envelope. Here we have determined the structure of the A33 ectodomain of vaccinia virus. The structure revealed C-type lectin-like domains (CTLDs) that occur as dimers in A33 crystals with five different crystal lattices. Comparison of the A33 dimer models shows that the A33 monomers have a degree of flexibility in position within the dimer. Structural comparisons show that the A33 monomer is a close match to the Link module class of CTLDs but that the A33 dimer is most similar to the natural killer (NK)-cell receptor class of CTLDs. Structural data on Link modules and NK-cell receptor-ligand complexes suggest a surface of A33 that could interact with viral or host ligands. The dimer interface is well conserved in all known A33 sequences, indicating an important role for the A33 dimer. The structure indicates how previously described A33 mutations disrupt protein folding and locates the positions of N-linked glycosylations and the epitope of a protective antibody.

FIG. 1.

A33 contains a dimer of C-type lectin-like domains (CTLDs). (A) A ribbon representation depicts the two monomers of A33, residues 98 to 185, on the membrane surface (“out”). The C100-C109 and C126-C180 disulfide bonds (orange) and the Asn side chains of the two N-linked glycosylation sites (magenta) are shown on each A33 CTLD monomer. The intermolecular disulfide bond between the C62 residues of each monomer and the palmitoylated C36 residues on the virion or cytosol side (“in”) of the membrane are shown. The β2 and β3 β-strands are labeled in panel B for clarity. The 36 residues from Cys 62 to the first ordered residue in the crystal, Glu 98, comprise the stalk between the membrane and A33. (B) View of A33 after a 90° rotation from the view in panel A. The predicted N-linked glycosylation sites (Asn 125 and Asn 135) are labeled. The three residues (166, 167, and 168) between residues 165 and 169 in both monomers were not modeled (see Methods).

FIG. 2.

Overlays of A33 and five other CTLD structures in stick representations. (A) A33 (red) is overlaid with five other CTLDs (blue and yellow, at left) and is shown alone (at right and in panel B) to orient the reader. The other CTLDs are colored blue where they exhibit the more conserved portions of a C-type lectin-like fold and are colored yellow where they are not as conserved. Conserved secondary structure elements are labeled from N to C termini: β1, α1, α2, β2, β3, β4, and β5. Note that A33 does not contain a β4 strand. The β2 strand of A33 (red) and the β2 strands of the other CTLDs (“β2,” colored yellow for clarity) also superimpose closely. The orientation here is similar to the orientation of the A33 monomer on the right in Fig. 1A. CTLDs that have been found to be homodimers were chosen for comparison. (B) The view is rotated toward the viewer and to the left about 45° in order to see the “long loop.” Each of the five CTLDs has the “long loop” connecting the β2 and β3 strands, but A33 does not. Instead, A33 has a short connecting loop (residues 155 to 157). The legend to Fig. 3 identifies the five structures from which monomer CTLDs were extracted and superimposed on the A33 monomer.

FIG. 3.

The A33 homodimer of CTLDs is structurally similar to CTLD dimers found in only a few other proteins. A ribbon depiction of the A33 dimer (Poxvirus A33) is shown at the upper left. Other known homodimers of CTLDs are from the immune system and are colored similarly for comparison: CD69 (PDB code 1e8i [46]), NKG2D (1kcg [59]), Ly49A (1qo3 [71]), CD94 (1b6e [5]), and Lox-1 (1ypo [56]). The KLRG1 dimer (3ff8 [45]) is also similar (not shown). The molecules are rainbow colored in a gradient from the N terminus (dark blue) to the C terminus (red). Disulfide bonds are shown in red. All proteins in this figure are type II transmembrane proteins.

FIG. 4.

The A33 dimer interface exhibits limited flexibility. The “B” monomers from each of the six A33 models were superimposed (red monomer at right). The relative positions of the “A” monomers at left (gray) are seen to vary among the six kinds of packing (see Table 2). The P2₁ crystal had two A33 dimers in the asymmetric unit. The A33 model from the “C2 (new)” crystal in which the A33 structure was determined is colored red.

FIG. 5.

The A33 dimer interface is conserved in the Chordopoxvirinae subfamily. (A) The top line is the sequence of A33 from vaccinia virus (VACV) in the structure described herein. The A33 sequence is aligned with the sequences of nine A33 orthologs: RFV (rabbit fibroma virus), TANV (tanapox virus), YMTV (yaba monkey tumor virus), LSDV (lumpy skin disease virus), SWPV (swinepox virus), DPV (deerpox virus), ORFV (orf virus), BPSV (bovine papular stomatitis virus), and MOCV (molluscum contagiosum virus). Sequence similarities have been shaded with the ClustalX color scheme. Cysteines for the C100-C109 and C126-C180 disulfides are completely conserved. (B) Surface representation of A33 with the molecule in the same view as in Fig. 1. The location of the putative ligand binding site is marked (*). The location of the surface implicated in the MAb 1G10 epitope and contributed by residues 118 and 120 is indicated on the “b” monomer (blue). Monomers “a” and “b” have been shaded red and blue, respectively, accordingly to the sequence conservation at that position in the alignment in panel A. A bright red or blue patch means that a high percentage of the sequences in panel A have the same residue as does vaccinia virus A33 at that position. The alignment in panel A was used as input to the ProtSkin server (12). Residues 118 and 120 of the “a” monomer (red) are not visible in this view. GenBank geninfo identifiers (gi) are as follows: VACV, 66275953; RFV, 9633931; TANV, 157939746; YMTV, 38229285; LSDV, 15150561; SWPV, 18640205; DPV, 62637509; ORFV, 32167503; BPSV, 41018861; MOCV, 9629074. The sequence of the squirrelpox virus A33 ortholog (SQV) (88769957) is not shown for clarity, due to an eight-residue insertion between 141 and 142 and a six-residue insertion between residues 160 and 161.