title: Design and analysis of an engineered human interleukin-10 monomer.
abstract: A monomeric form of human interleukin 10 (IL-10M1) has been engineered for detailed structure-function studies on IL-10 and its receptor complexes. Wild type IL-10 (wtIL-10) is a domain swapped dimer whose structural integrity 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 the swapped secondary structural elements. Characterization of IL-10M1 by mass spectroscopy, size exclusion chromatography, cross-linking, and circular dichroism shows that IL-10M1 is a stable alpha-helical monomer at physiological 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-10Ralpha (sIL-10Ralpha) generates a complex that consists of 2 wtIL-10 molecules and 4 sIL-10Ralphas. In contrast, IL-10M1 forms a 1:1 complex with the sIL-10Ralpha. Characterization of the interaction using isothermal 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 interaction between IL-10M1 and IL-10Ralpha is sufficient for recruiting the signal transducing receptor chain (IL-10Rbeta) into the signaling complex and eliciting IL-10 cellular responses.
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
authors.forename: K
authors.initials: K
authors.lastname: Josephson
authors.forename: R
authors.initials: R
authors.lastname: DiGiacomo
authors.forename: S R
authors.initials: SR
authors.lastname: Indelicato
authors.forename: A H
authors.initials: AH
authors.lastname: Iyo
authors.forename: T L
authors.initials: TL
authors.lastname: Nagabhushan
authors.forename: M H
authors.initials: MH
authors.lastname: Parker
authors.forename: M R
authors.initials: MR
authors.lastname: Walter
authors.forename: A H
authors.initials: AH
authors.lastname: Ayo
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D019281:Dimerization; D015195:Drug Design; D005818:Genetic Engineering; D006801:Humans; D016753:Interleukin-10 / Q000032:analysis* / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D011487:Protein Conformation
chemical_list: D016753:Interleukin-10