Variola virus IL-18 binding protein interacts with three human IL-18 residues that are part of a binding site for human IL-18 receptor alpha subunit

Abstract

Interleukin-18 (IL-18) plays an important role in host defense against microbial pathogens. Many poxviruses encode homologous IL-18 binding proteins (IL-18BP) that neutralize IL-18 activity. Here, we examined whether IL-18BP neutralizes IL-18 activity by binding to the same region of IL-18 where IL-18 receptor (IL-18R) binds. We introduced alanine substitutions to known receptor binding sites of human IL-18 and found that only the substitution of Leu5 reduced the binding affinity of IL-18 with IL-18BP of variola virus (varvIL-18BP) by more than 4-fold. The substitutions of Lys53 and Ser55, which were not previously known to be part of the receptor binding site but that are spatially adjacent to Leu5, reduced the binding affinity to varvIL-18BP by approximately 100- and 7-fold, respectively. These two substitutions also reduced the binding affinity with human IL-18R alpha subunit (hIL-18Ralpha) by 4- and 2-fold, respectively. Altogether, our data show that varvIL-18BP prevents IL-18 from binding to IL-18R by interacting with three residues that are part of the binding site for hIL-18Ralpha.

Structural basis for antagonism of human interleukin 18 by poxvirus interleukin 18-binding protein

“…Lys-53 also forms a hydrogen bond and a salt bridge, respectively, with Glu-69 and Glu-77 of ectvIL-18BP, effectively lodging hIL-18 Lys-53 into a remarkably secure position. These intimate interactions form a network of stabilizing forces at binding site A, fully explaining some previous mutagenesis studies on various IL-18BPs and hIL-18 (14)(15)(16)(17). These studies identified Tyr-53 and Phe-67 of ectvIL-18BP and Lys-53 of hIL-18 as residues that contribute most significantly to complex formation between these 2 molecules.…”

“…The current crystal structure displays a 1,930-Å 2 buried solventaccessible area that is much larger than that observed in the IL-1␤-IL-1R␣ complex at site II (1,000 Å 2 ) (18). Assuming that hIL-18 binds its receptor ␣-subunit in mode similar to that observed in the IL-1␤-IL-1R␣ complex, our structure suggests a much tighter binding by IL-18BP compared with its receptor, which is further supported by binding studies (8,15,16). Therefore, the current structure supports a general inhibition mechanism by which IL-18BP neutralizes hIL-18 through competition with IL-18R␣ at a common binding site, thus antagonizing IL-18 and mitigating its effects on IFN-␥ production and host immune response.…”

“…The current complex structure identifies Ϸ25 hIL-18 residues that are involved in ectvIL-18BP binding ( Table 1). Six of these residues (Leu-5, Lys-8, Lys-53, Arg-58, Met-60, and Arg-104) were suggested to be part of site II that is involved in IL-18R␣ binding (12,16). Therefore, ectvIL-18BP predominantly blocks the accessibility of the receptor at the putative binding site II on hIL-18 (Fig.…”

“…Sites I and II were suggested to be involved in IL-18R␣ binding, whereas site III was proposed to bind IL-18R␤. Variola virus IL-18BP has been shown to interact with 3 hIL-18 residues that are part of the site II (16). Human IL-18BP was predicted to interact with the same site based on homology modeling studies (19), therefore suggesting that all IL-18BPs compete with IL-18R␣ for site II of hIL-18.…”

Vaccinia Virus A6L Encodes a Virion Core Protein Required for Formation of Mature Virion

“…The V5 coding sequences in the primers are underlined. The recombinant PCR product was cloned into the EcoRI and AccI sites of pYW31 (14) flanking the green fluorescent protein (gfp) and xanthine-guanine phosphoribosyltransferase (gpt) cassettes.…”