Technical Highlight - June 2013
Short description: Crystal structures of cytokine receptor complexes pinpoint target regions to create 'superkines' with altered cell specificity.
Cytokines orchestrate wide-ranging signaling cascades in cells. Cytokine receptor activation is initiated by interaction with one receptor chain followed by a second. The second interaction is of much lower affinity, so its manipulation is considered promising for potential therapeutic applications; this is the interaction Garcia and colleagues targeted in their study of interleukin-4 (IL-4). IL-4 first binds the receptor IL-4Rα with high affinity; a subsequent interaction with either γc or IL-13Rα1 results in, respectively, type I and type II ternary complexes. Secondary cytokine receptors are expressed at different levels in various cells, thus enabling cell-type specific signaling.
Recently solved crystal structures reveal how γc and IL-13Rα1 bind IL-4. To alter affinity to type II receptors, the authors compared structures of IL-4 and IL-13 bound to IL-13Rα1, which led them to engineer a high-affinity version of IL-4 by substituting three residues from the native high-affinity binder IL-13. With no such information for the type I receptor, the researchers used in vivo evolution. These respective approaches resulted in the development of high-affinity triple mutant 'superkines' KFR and RGA.
In vitro, as measured by surface plasmon resonance (SPR), binding affinity of KFR–IL-4Rα for the type II receptor IL-13Rα1 was 440-fold greater than that of wild-type IL-4–IL-4Rα, and for the type I receptor γc, about twofold lower. In contrast, binding affinity of RGA–IL-4Rα for its destined receptor γc was 3,700-fold greater than that of wild-type cytokine complex, also with a decrease in affinity to IL-13Rα1. Structural analysis of the RGA–γc interface (PDB 3QB7) pinpointed changes responsible for the affinity increase.
This binding specificity was preserved in vivo, in cells that naturally express different amounts of type I and type II receptors. The differences, however, were not as dramatic as those observed by SPR, which the authors attribute to differences in diffusion rates when one of the components is soluble in the latter setup, or to co-localization of receptors in vivo. Nevertheless, this proof-of-principle work suggests the possibility of engineering, for therapeutic applications, superkines with high affinities to target receptors and those with minimal affinity to other receptors, thus limiting side effects.
I.S. Junttila et al. Redirecting cell-type specific cytokine responses with engineered interleukin-4 superkines.
Nat. Chem. Biol. 8, 990-998 (2012). doi:10.1038/nchembio.1096