Design and analysis of an engineered human interleukin-10 monomer

A monomeric form of human interleukin 10 (IL-10M1) has been engineered for detailed structure-function studies on IL-10 and its receptor complexes. Wi ld type IL-10 (wtIL-10) is a domain swapped dimer whose structural integrit y depends on the intertwining of two peptide chains. wtIL-10 was converted to a monomeric isomer by inserting 6 amino acids into the loop connecting t he swapped secondary structural elements. Characterization of IL-10M1 by ma ss spectroscopy, size exclusion chromatography, cross-linking, and circular dichroism shows that IL-10M1 is a stable alpha-helical monomer at physiolo gical pH whose three-dimensional structure closely resembles one domain of wtIL-10. As previously reported, incubation of wtIL-10 with a soluble form of the IL-10R alpha (sIL-10R alpha) generates a complex that consists of 2 wtIL-10 molecules and 4 sIL-10R alpha s. In contrast, IL-10M1 forms a 1:1 c omplex with the sIL-10R alpha. Characterization of the interaction using is othermal titration calorimetry confirmed the 1:1 stoichiometry and yielded a dissociation constant of 30 nM with an apparent binding enthalpy of -12.2 kcal/mol. Despite forming a 1:1 complex, IL-10M1 is biologically active in cellular proliferation assays. These results indicate that the 1:1 interac tion between IL-10M1 and IL-10R alpha is sufficient for recruiting the sign al transducing receptor chain (IL-10R beta) into the signaling complex and eliciting IL-10 cellular responses.