A Unique Bivalent Binding and Inhibition Mechanism by the Yatapoxvirus Interleukin 18 Binding Protein

Abstract

Interleukin 18 (IL18) is a cytokine that plays an important role in inflammation as well as host defense against microbes. Mammals encode a soluble inhibitor of IL18 termed IL18 binding protein (IL18BP) that modulates IL18 activity through a negative feedback mechanism. Many poxviruses encode homologous IL18BPs, which contribute to virulence. Previous structural and functional studies on IL18 and IL18BPs revealed an essential binding hot spot involving a lysine on IL18 and two aromatic residues on IL18BPs. The aromatic residues are conserved among the very diverse mammalian and poxviruses IL18BPs with the notable exception of yatapoxvirus IL18BPs, which lack a critical phenylalanine residue. To understand the mechanism by which yatapoxvirus IL18BPs neutralize IL18, we solved the crystal structure of the Yaba-Like Disease Virus (YLDV) IL18BP and IL18 complex at 1.75 Å resolution. YLDV-IL18BP forms a disulfide bonded homo-dimer engaging IL18 in a 2∶2 stoichiometry, in contrast to the 1∶1 complex of ectromelia virus (ECTV) IL18BP and IL18. Disruption of the dimer interface resulted in a functional monomer, however with a 3-fold decrease in binding affinity. The overall architecture of the YLDV-IL18BP:IL18 complex is similar to that observed in the ECTV-IL18BP:IL18 complex, despite lacking the critical lysine-phenylalanine interaction. Through structural and mutagenesis studies, contact residues that are unique to the YLDV-IL18BP:IL18 binding interface were identified, including Q67, P116 of YLDV-IL18BP and Y1, S105 and D110 of IL18. Overall, our studies show that YLDV-IL18BP 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 IL18. However, despite this extensive divergence, YLDV-IL18BP binds to the same surface of IL18 used by other IL18BPs, suggesting that all IL18BPs use a conserved inhibitory mechanism by blocking a putative receptor-binding site on IL18.

Crystallization of interleukin-18 for structure-based inhibitor design

“…Notice that the mutation sites are distant from the IL-18BP and IL-18R D3 domain interfaces. Krumm et al, 2008Krumm et al, , 2012. As observed previously in the viral inhibitory complexes, Lys53 of binding site A in hIL-18 adopts a conserved interaction at the binding interface with the receptor.…”

“…The first structure of hIL-18 was solved by solution NMR and other structures of hIL-18 have been determined either in complex with its cognate receptor(s), IL-18BPs or with hIL-18-specific antibodies (Kato et al, 2003;Krumm et al, 2008Krumm et al, , 2012Wei et al, 2014;Argiriadi et al, 2009;Tsutsumi et al, 2014). Apo hIL-18 has been recalcitrant to crystallization.…”

“…As an immune-evasion strategy, homologs of IL-18BPs are also encoded by many poxviruses such as Molluscum contagiosum virus, orthopoxviruses such as Variola virus (the causative agent of smallpox) and yatapoxviruses such as Yaba-like disease virus (YLDV). The recent crystal structures of hIL-18 in complex with Ectromelia virus (ectv; Krumm et al, 2008) and YLDV (Krumm et al, 2012) IL-18BP revealed a conserved mechanism of hIL-18 inhibition through direct competition for a common binding site with the hIL-18 primary receptor (hIL-18R) domain 3 (D3; Tsutsumi et al, 2014;Wei et al, 2014).…”

“…The crystal structures revealed a pliable surface of hIL-18 that undergoes an induced-fit mechanism upon binding IL-18BPs, involving three ligand-binding sites (A, B and C) on hIL-18 (Krumm et al, 2008(Krumm et al, , 2012Figs. 1a and 1b).…”

“…Contained within binding site A is a critical hIL-18 lysine residue (Lys53), which when mutated to alanine displayed a greater than 100-fold and fourfold decrease in binding affinity for Variola virus IL-18BP and hIL-18R, respectively (Meng et al, 2007), indicating that binding site A on the hIL-18 surface is a 'hot spot' that is critical not only for its interaction with viral IL-18BPs but also for initial hIL-18-receptor complex formation. Additionally, binding site C on the hIL-18 interface is another potential 'hot spot' as it contains a conserved phenylalanine residue that has been shown to be critical for complex formation with orthopoxviruses, Molluscum contagiosum virus (MCV) and human IL-18BP (Esteban & Buller, 2004;Krumm et al, 2012;Xiang & Moss, 2001a,b).…”