Structural and functional studies on the modulation of interleukin-18 activity

Interleukin-18 (IL-18) is a pleiotropic pro-inflammatory cytokine that plays an important role in inflammation as well as in the innate and adaptive immune defense through stimulation of Interferon-gamma (IFN-gamma) production. Its activity is regulated in vivo by a naturally occurring antagonist, the human IL-18 Binding Protein (IL-18BP). Functional homologs of human IL-18BP are encoded by all orthopoxviruses, including variola virus, the causative agent of smallpox, and contribute to their virulence by suppressing IL-18 mediated immune response. In this work, we have focused on the structural and functional characterization of human IL-18 in complex with IL-18BP from two distinct species of poxviruses, the ectromelia virus (ECTV) and the Yaba Like Disease Virus (YLDV), using biophysical methods including X-ray crystallography. Through virtual screening and cell-based bioassays, we have also identified small molecules that specifically target hIL-18 and modulate its bioactivity. Here, we describe the first three-dimensional structure of an orthopoxvirus IL-18BP, ECTV IL-18BP, in complex with hIL-18 in a 1:1 stoichiometry to 2.0A resolution. The hIL-18 structure in the complex shows significant conformational change when compared to the ligand-free hIL-18 NMR solution structure, indicating the binding is mediated via an induced-fit mechanism. ECTV IL-18BP adopts a canonical h-type immunoglobulin-fold and interacts via one edge of its beta-sandwich with three induced cavities on the hIL-18 surface, using extensive hydrophobic and hydrogen bonding interactions through conserved residues found in mammalian and orthopoxvirus IL-18BPs. We also determined the three-dimensional structure of a yatapoxvirus IL-18BP, YLDV IL-18BP, in complex with hIL-18 to 1.75A resolution. Strikingly, YLDV IL-18BP forms a unique disulfide linked homodimer and binds hIL-18 in a 2:2 stoichiometry. Each protomer complex resembles the ECTV IL-18BP:hIL-18 complex while also using an induced fit mechanism to bind hIL-18. Our studies reveal YLDV IL-18BP is unique among the diverse family of mammalian and poxvirus IL-18BPs in that it uses a bivalent binding mode and a unique set of interacting residues for binding hIL-18. Our studies also reveal poxvirus family members neutralize hIL-18 activity with a similar induced fit mechanism despite extensive divergence, suggesting that all IL18BPs use a conserved inhibitory mechanism by blocking a putative receptor-binding site on hIL18.