Volume 9, Issue 2, Pages 375-385 (February 2002) Structure of the Epstein-Barr Virus gp42 Protein Bound to the MHC Class II Receptor HLA-DR1  Maureen M. Mullen, Keith M. Haan, Richard Longnecker, Theodore S. Jardetzky  Molecular Cell  Volume 9, Issue 2, Pages 375-385 (February 2002) DOI: 10.1016/S1097-2765(02)00465-3

Figure 1 Structure of EBV gp42 (A) Initial electron density map for gp42 calculated with model phases from the DR1:HA molecular replacement solution followed by density modification with CNS (Brunger et al., 1998). (B) Stereo view of the Cα trace of the gp42 as observed in the gp42:DR1 complex. Side chains for the gp42 disulfide bonds and potential N-linked glycosylation sites are shown. (C) Schematic diagram of the ten cysteines that form five disulfide bonds in gp42. Two are common within the C-type lectins, two are analogous to disulfide bonds observed in the Ly49 and CD94 NK receptors, and one is unique to the gp42 fold, pinning down the N-terminal region to the core CTLD. Molecular Cell 2002 9, 375-385DOI: (10.1016/S1097-2765(02)00465-3)

Figure 2 Sequence Alignment of gp42 and NK Receptors Sequences of gp42, Ly49A, CD94, and NKG2D are compared. The core secondary structure elements of the gp42 CTLD are indicated above the sequence alignment. Four strands are indicated (βD, β1′, β2′, and β2′′) that are not part of the standard CTLD core structure. The βD strand forms dimer contacts in the crystal and the β1′ strand, although present in other CTLD structures, is short (3 residues). The β2′ and β2′′ form a short, two-stranded sheet on the outer edge of the CTLD. Cysteines that form disulfide bonds are highlighted in yellow, and completely conserved residues are highlighted in blue. Disulfide bonds are indicated by blue lines. Residues within 4 Å of HLA-DR1 atoms in the complex are indicated by red dots above the gp42 sequence, and hydrophobic residues located in the CTLD canonical binding site are indicated by magenta dots. Molecular Cell 2002 9, 375-385DOI: (10.1016/S1097-2765(02)00465-3)

Figure 3 Interactions between gp42 and HLA-DR1 (A) Ribbon diagram of the gp42:DR1 complex. gp42 is shown in red, DR1:HA in blue. The N-terminal region of gp42 forms a two-stranded, antiparallel β sheet with a crystallographically related symmetry mate. (B) Interactions formed by DR1 Eβ46 with gp42 residues. The gp42 chain is shown in red and DR1 in blue. Eβ46 forms a salt bridge (R220) and a hydrogen bond (Y107) across the interface, and single point mutations of Eβ46 have been shown to affect EBV entry and gp42 binding. (C) Interactions formed by DR1 Rβ72 with gp42 residues. Chains are colored as in (B). Rβ72 interacts primarily with the backbone atoms of the turn before the first β strand of the gp42 CTLD, including residues 104–107 and with the side chain of Y107. gp42 residues T104 and Y107 interact with both Eβ46 and Rβ72. Molecular Cell 2002 9, 375-385DOI: (10.1016/S1097-2765(02)00465-3)

Figure 4 Comparison of the gp42:DR1 Complex to Ly49A and NKG2D NK Receptor Complexes (A) Surface of gp42 involved in MHC class II binding. The complete surface of gp42 is shown along with the DR1 structure (right), with the binding site colored blue. The orientation of gp42 is shown to the left, overlayed with the DR1 binding surface. (B) The surface of one of the Ly49A monomers involved in dimerization and binding the MHC class I molecule H-2Dd (right). The protein interaction surfaces are shown in blue. The orientation of this Ly49A monomer is similar to the gp42 in (A) as shown in the left panel, with the interaction surfaces overlayed onto the Cα trace. Note that the dimerization surface of Ly49A corresponds to the gp42 surface involved in MHC class II interactions, as indicated. (C) The surface of one of the NKG2D monomers involved in dimerization and binding to the MHC class I homolog MICA (right). The orientation of this NKG2D monomer is similar to both the gp42 and Ly49A shown in (A) and (B), respectively. Note that the NKG2D and Ly49A monomers use similar regions of the CTLD fold in both dimerization and ligand binding interactions, although these two proteins have limited homology. The ligand binding region is similar to the carbohydrate binding region observed in lectins of this superfamily, defining a “canonical ligand binding site.” gp42 binding to DR1 involves a surface analogous to the dimerization surface of these NK receptors, leaving the “canonical” binding site free for interactions with other potential ligands. Molecular Cell 2002 9, 375-385DOI: (10.1016/S1097-2765(02)00465-3)

Figure 5 Hydrophobic Character of the gp42 Canonical Ligand Binding Pocket (A) The gp42 structure reveals that a number of surface-exposed aromatic and hydrophobic residues line the putative ligand binding site. Aromatic residues F188, F198, F210, and Y185 are exposed at surface loops lining this site, with Y194 located at the base of the site. In addition, aliphatic residues V184, I159, I187, V201, and L211 contribute to additional hydrophobic character of the surface loops. (B) Surface representation of the gp42:DR1 complex showing the location of the aromatic and hydrophobic residues in (A). The putative binding site forms a broad and shallow groove that is oriented up and away from the MHC class II molecule, potentially directed away from the target cell membrane and toward the virus surface. (C) Hydrophobic surface patches identified by the program Quilt (Lijnzaad et al., 1996). Quilt was used to analyze the gp42 monomer, and the two largest hydrophobic patches are displayed. Atoms contributing to the largest hydrophobic patch (∼620 Å2) are shown in red. Atoms contributing to the second largest patch (∼396 Å2) are shown in blue. The top two hydrophobic patches define two of the three sides to the canonical binding site surface in gp42. Molecular Cell 2002 9, 375-385DOI: (10.1016/S1097-2765(02)00465-3)

Figure 6 Dimerization of gp42 and HLA-DR1 in the Crystal (A) Side and (B) top views of gp42 dimers observed in the crystal. The N-terminal residues of gp42 (87–94) form an anti-parallel β sheet in the crystal lattice, allowing a crossover between adjacent gp42:DR1 complexes. The N terminus of each gp42 molecule is indicated by the yellow circle (residue 86), and arrows point toward the canonical CTLD binding site. The orientation of the DR1 molecules is consistent with such an interaction occurring at the cell surface. The dimerization places the disordered gp42 residues (33–85) implicated in binding to gH/gL onto neighboring gp42:DR1 complexes, near the canonical ligand binding site. Formation of dimers may enhance additional interactions of gp42 with gH/gL, or the recruitment of gB or other ligands into a larger triggering complex. Molecular Cell 2002 9, 375-385DOI: (10.1016/S1097-2765(02)00465-3)