Deciphering Site 3 Interactions of Interleukin 12 and Interleukin 23 with Their Cognate Murine and Human Receptors

Interleukin (IL)-12 and IL-23 belong to the IL-12 type family and are composite cytokines, consisting of the common β subunit p40 and the specific cytokine α subunit p35 and p19, respectively. IL-12 signals via the IL-12Rβ1·IL-12Rβ2 receptor complex, and IL-23 uses also IL-12Rβ1 but engages IL-23R as second receptor. Importantly, binding of IL-12 and IL-23 to IL-12Rβ1 is mediated by p40, and binding to IL-12Rβ2 and IL-23R is mediated by p35 and p19, respectively. Previously, we have identified a W157A substitution at site 3 of murine IL-23p19 that abrogates binding to murine IL-23R. Here, we demonstrate that the analogous Y185R site 3 substitution in murine and Y189R site 3 substitution in human IL-12p35 abolishes binding to IL-12Rβ2 in a cross-species manner. Although Trp is conserved between murine and human IL-23p19 (Trp in the human ortholog), the site 3 W156A substitution in hIL-23p19 did not affect signaling of cells expressing human IL-12Rβ1 and IL-23R, suggesting that the interface of murine IL-23p19 required for binding to IL-23R is different from that in the human ortholog. Hence, we introduced additional hIL-23p19 substitutions within its binding interface to hIL-23R and found that the combined site 3 substitutions of W156A and L160E, which become buried at the complex interface, disrupt binding of hIL-23p19 to hIL-23R. In summary, we have identified substitutions in IL-12p35 and IL-23p19 that disrupt binding to their cognate receptors IL-12Rβ2 and IL-23R in a murine/human cross-species manner.