We previously determined the crystal structure of the ectromelia virus IL18BP in complex with IL18, revealing the structural basis for the binding and inhibition of IL18 by the IL18BPs 
. Despite an overall low sequence homology between the diverse viral and host IL18BPs, the key residues of ECTV IL18BPs at the IL18 binding interface are highly conserved. Mutagenesis studies on human and viral IL18BPs also showed that these key residues are almost universally critical for the binding of IL18BPs to IL18. Thus it was enigmatic that functional yatapoxvirus IL18BPs lack a key phenylalanine residue () that has been identified to be essential for many other IL18BPs to bind IL18 
. It is similarly puzzling that a residue of IL18 (K53) that is critical for binding orthopoxvirus IL18BPs only played a modest role in binding with the YMTV-IL18BP 
. In this report, we resolved these questions by determining the crystal structure of YLDV-IL18BP:IL18 complex and by performing extensive mutagenesis and SPR studies. We revealed two unique signature features of YLDV-IL18BP that distinguish yatapoxvirus IL18BPs from the rest of IL18BP family members. First, YLDV-IL18BP forms a homo-dimer and interacts with IL18 in a 2
2 binding mode. Second, the binding of YLDV-IL18BP and IL18 does not rely on two of the ‘hot spot’ interactions that were shown to be essential for the binding of all previously studied IL18BPs, including a phenylalanine (F67 in ECTV-IL18BP) at site A and another phenylalanine at site C (F49 in ECTV-IL18BP). Instead, yatapoxvirus IL18BPs evolved interactions with some IL18 residues (Y1, D110, S105) that are specifically important for binding with YLDV-IL18BP. It appears that YLDV-IL18BP shifts and disperses the binding energy across the IL18-binding interface rather than concentrating the binding energy on a few hot spots as is the case for all other IL18BPs examined to date.
It was previously reported that ECTV-IL18BP and human IL18BP are monomeric in solution 
. In contrast, YLDV-IL18BP forms a disulfide-bonded dimer, which was demonstrated not only in the crystal structure but also in solution by non-reducing SDS-PAGE and gel filtration analysis. The dimer interface is quite large (about 1,700 Å2
) and involves extensive hydrophobic interactions in addition to the intermolecular disulfide bond, indicating that the YLDV-IL18BP dimer is intrinsically stable in solution. The dimer could only be separated into monomers by mutations that disrupt both the hydrophobic interactions as well as the inter-chain SS bond. Analysis of the monomeric YLDV-IL18BP (HVEC) showed that the dimerization was not essential for binding IL18 but enhanced the binding affinity by 3-fold in our in vitro
assay. Although this enhancement in binding affinity as measured by SPR is modest, it is possible that the dimerization may be more important for the function of YLDV-IL18BP during infection of the host, perhaps by increasing the half-life of the protein in the infected tissue or by increasing the avidity of binding to IL18 at low protein concentration. In fact, divalent or multivalent binding is an important, inherent feature of many biological systems to enhance the effectiveness of binding of ligands to receptors and of antibodies to antigens 
. More specifically, this has been a feature for quite a few poxvirus cytokine binding proteins. For example, ectromelia virus IFN-γ binding protein forms a tetramer, which is required for efficient IFN-γ antagonism 
. Myxoma virus T2 protein, a Tumor Necrosis Factor (TNF) Receptor homolog, is secreted as both monomer and dimer, and the dimeric T2 is a more potent TNF inhibitor 
. Because residues of YLDV-IL18BP involved in dimer formation are only conserved in yatapoxviruses, yatapoxviruses IL18BPs may be unique among IL18BPs in that they use bivalent binding to increase the affinity and avidity for IL18.
Another difference between YLDV-IL18BP and all other IL18BPs is the lack of two of the ‘hot spot’ interactions at the binding sites A and C on the surface of IL18. The structure of ECTV-IL18BP:IL18 complex showed that a conserved phenylalanine (F67) is engaged in hydrophobic and strong π-cation interactions with K53 of IL18 at binding site A 
. Alanine substitutions of K53 of IL18 significant decreased binding with orthopoxvirus IL18BPs 
, while alanine substitutions of the conserved phenylalanine (equivalent to ECTV-IL18BP F67) in human, MCV and orthopoxviruses IL18BPs significantly decreased or completely abolished binding of IL18 
. The current structure of YLDV-IL18BP:IL18 complex showed that a threonine residue (T64) is present at the position equivalent to the phenylalanine, indicating the π-cation interaction with K53 is not important for YLDV-IL18BP: IL18 complex. Indeed, T64F or T64A substitution of YLDV-IL18BP had negligible or minor (2.5-fold decrease in affinity for T64A, student t-test P-value<0.05) effect on the binding with IL18, while K53A of IL18 had a more modest effect on binding with YLDV-IL18BP than with orthopoxvirus IL18BPs. A similar loss of ‘hot spot’ interaction was also observed at binding site C. A phenylalanine residue on IL18BPs that binds to site C of IL18 was previously shown to be important for binding IL18 in orthopoxvirus, MCV and human IL18BPs 
. Although a phenylalanine (F52) is present at the equivalent position in YLDV-IL18BP, it is not important for binding to IL18 (, ,). Through structural and mutagenesis studies, we have identified contact residues that are unique to the YLDV-IL18BP:IL18 binding interface. This includes Q67 of YLDV-IL18BP and Y1 of IL18 at site A, P116 of YLDV-IL18BP and S105 and D110 of IL18 at site C. Our data are in agreement with the conclusion of a more delocalized energy distribution for binding of IL18 to YMTV-IL18BP 
. The structural and functional studies of two different IL18BP complexes suggest that there is a degree of plasticity in the IL18BP:IL18 interface that could accommodate certain mutations in IL18BPs without compromising their binding affinity to IL18.
Despite the differences in several key residues for binding IL18, the current YLDV-IL18BP:IL18 complex structure showed that YLDV-IL18BP targets the same surface of IL18 as ECTV-IL18BP does in the previous complex structure. This suggests that all IL18BPs inhibit IL18 function by blocking a putative receptor-binding site on the surface of IL18. Similar to previous findings on human and poxvirus IL18BPs, Y56 of YLDV-IL18BP (interacting with site A of IL18) was found to be absolutely essential for binding to IL18, indicating that this conserved tyrosine residue is an obligatory ‘anchor’ for binding of all IL18BPs to IL18. The conservation and variation in functional residues and their specific interactions with IL18 suggest that IL18BPs share a common ancestor but may have undergone significant evolution through different selection pressures, resulting in a conserved inhibitory mechanism albeit with mutations of interface residues.
The biological activity of IL18 is determined in part by its relative affinities for IL18 receptors and IL18BP. The binding of IL18 to its receptors triggers multiple cellular responses vital to immunity, but excessive IL18 activities are associated with many autoimmune and inflammatory diseases 
. Functional IL18BPs are present in many poxviruses including variola virus and vaccinia virus, providing a key strategy of poxvirus immune evasion by inhibition of IL18 cytokine activity. Therefore the studies on IL18BP:IL18 inhibitory complexes could serve dual purposes by providing important clues on how to develop functional inhibitors targeting either IL18 or poxvirus IL18BP. These inhibitors could potentially modulate IL18 and poxvirus IL18BP activities, which may benefit efforts in developing treatments against some autoimmune and inflammatory diseases and in developing treatments for potential pathogenic outbreaks associated with poxvirus infections.