Table 1.
Binding kinetics measured by SPR.
Figure 1.
Crystal structure of the UL141–TRAIL-R2 complex.
(a) Hetero-tetrameric structure of the UL141 dimer (orange and cyan cartoon) in complex with two TRAIL-R2 monomers (grey surface). Six distinct binding patches between UL141 and TRAIL-R2 are indicated with dotted circles. (b) 2-D topology diagram (generated by PDBsum [56]) of the UL141 subunit with arrows from N-terminus to C-terminus and two cartoon representations of one UL141 subunit 90° rotated. The N-terminal domain of UL141 exhibits a V-type immunoglobulin superfamily fold containing ten β-strands (a–g) and two short α-helices H1 and H2 (orange). The C-terminal domain contains three β-strands (1–3) and two short α-helices H3 and H4 (light orange). Disulfide bonds (C84–C234 and C67–C143) are indicated as yellow sticks. Potential N-linked asparagines are drawn as cyan sticks and glycans are shown in grey for one UL141 monomer (chain A) at position N132 and N147, while N117 is occupied in the other monomer (chain B). Disordered loops 168–174, 199–207 and 217–226 are indicated as dotted lines. (c) The cartoon representation of TRAIL-R2 structure colored green with molecular surface in grey. Dotted lines delineate CRDs (Cysteine Reach Domains); CRD-1 (residues 78–94), CRD-2 (residues 95–137) and CRD-3 (residues 138–178). Disulfide bonds are depicted in yellow as ball-and-sticks. The β1β2 loop of CRD-3 (residues 143–157) and that of CRD-2 (residues 104–115) that make the key contacts with the ligands as well as other important loops (β5β6 of CRD-2, β2β3 of CRD-3, N-term loop) and CXC motif are highlighted by circle or arrows.
Table 2.
Data collection and refinement statistics.
Figure 2.
Comparison of the UL141 and TRAIL binding footprints on TRAIL-R2.
TRAIL-R2 is shown as a grey molecular surface in three different orientations: left (a), front (b), and right (c). The binding interface between the UL141 and TRAIL-R2 (c) is divided into six binding patches, with patches 3 and 5 being similar to that of the TRAIL–TRAIL-R2 complex (a). TRAIL contact residues on TRAIL-R2 in cyan (a–b), UL141 contact residues on TRAIL-R2 in yellow (b–c), while the overlapping residues are green (b).
Figure 3.
Comparison of TRAIL-R2 structures.
The TRAIL-R2 structure derived from the complex with UL141 (4I9X, in cyan) is superimposed (residues 78–178) with available TRAIL-R2 crystal structures from PDB database. Three TRAIL-R2–TRAIL structures: 1D4V (grey), 1DU3 (green) and 1D0G (light purple) and two TRAIL-R2–Fab structures: YSd1 Fab (1Z3A, red) and BdF1 Fab (2H9G, yellow). (a) Structures superimpose very well with the exception of the β1β2 loop of CRD-3 (Patch 3). Representative 2FO-FC electron density map contoured at 1σ, showing the key residues of receptor β1β2 loop (cyan) interacting with UL141 residues (orange) in patch 3 (b) and patch 3 U (c) in stereo views. The well-defined electron density indicates, that the β1β2 loop of CRD-3 is well ordered upon UL141 binding.
Figure 4.
Comparison of receptor-ligand interaction between UL141 and TRAIL with TRAIL-R2 and mutational binding data.
(a) Detailed interaction is shown for the six binding patches of the UL141–TRAIL-R2 complex; 1–2 (yellow), 3 (green), 3 U (light-green), 4 (pink), 5 (red), 6 (orange), as well as for the patches 3, 3T (blue), 4 and 5 of the TRAIL–TRAIL-R2 complex (same coloring scheme). Interaction residues are labeled and drawn as orange (UL141), salmon (TRAIL) and cyan (TRAIL-R2) sticks with atoms colored as follows: nitrogen (blue), oxygen (red) and sulfur (yellow). The molecular contacts (hydrogen bonds and salt bridges with distance <4.0 Å) are shown as dashed black lines. The name of interacting loop, helix or strand is listed around each box as well as the specificity of particular contact patch. (b) Open book view of UL141–TRAIL-R2 complex with their molecular surfaces outlined in grey. All binding patches (fingerprints on both molecules) follow the same color-code as above including residues selected for alanine scanning mutagenesis. (c) Relative effect on alanine mutagenesis of TRAIL-R2 on UL141 (middle column) and TRAIL (right column) binding, as analyzed by SPR (Figure 5 and Table S1). Mutated residues are listed (left column). Mutation that do not affect receptor binding are labeled ‘YES’ while ‘NO’ indicates binding is abrogated. X-fold reduction in binding (compared to wild-type) is quantitated by numbered arrows.
Figure 5.
Binding of TRAIL-R2 mutants to TRAIL and UL141.
Surface plasmon resonance study to assess the binding contribution of individual TRAIL-R2 residues to both viral UL141 and endogenous TRAIL. A Sensorgram for each kinetics experiment is shown in colored boxes (colored by binding patch as in Figure 4). The specific alanine mutation on TRAIL-R2 as well as the calculated binding constant KD (nM) are indicated for each panel. Mutations that fully abrogate binding are indicated as n.d. (binding not detected).
Figure 6.
UL141 surface accessibility for receptor binding.
Structure of one UL141 subunit (colored surface) in complex with one TRAIL-R2 (grey cartoon) shown in two views (0° and 180° turn). All three potential N-linked glycosylation sites (Asn117, Asn132 and Asn147) where modeled with a five-sugar Man2GluNac2Fuc glycans, shown in dark grey ball-and-stick). Area A and B indicate available and accessible protein binding sites on UL141, while other available areas are expected to be mostly covered with glycans in the fully glycosylated protein. Location of potential protein-protein binding sites for unbound UL141 were calculated using ProMate (http://bioinfo.weizmann.ac.il/promate). For simplicity, only one UL141 subunit is shown here in molecular surface colored from orange reflecting the lowest probability assigned, to green, assigned to the highest probability. The highest probability areas that reflect possible binding sites in UL141; excluding those binding sites 1–6 of TRAIL-R2 (shown in dotted line here); and are not shield by glycans, are areas A and B.