Analysis of interleukin-10 and its interaction with interleukin-10 receptor-1 by x-ray crystallography and protein engineering.

摘要

Interleukin-10 (IL-10) is a helical cytokine that controls and eventually terminates inflammatory responses by inhibiting the production of numerous pro-inflammatory cytokines and cell surface proteins. IL-10 activates the intracellular signal transduction pathways that ultimately lead to all of these cellular responses by organizing Interleukin-10 Receptor 1 (IL-10R1) and Interleukin-10 Receptor 2 (IL-10R2) in a cell surface cytokine receptor complex.; IL-10 is a symmetric homodimer, which folds as two identical helical bundle domains. In solution, IL-10 and a soluble form of IL-10RI (sIL-10R1) form a stable complex comprised of two IL-10 dimers and four sIL-10RI chains. We determined the crystal structure of this 2:4 IL-10/sIL-10R1 complex in order to understand its architecture and define the interactions that mediate its assembly. The structure revealed that the 2:4 IL-10/sIL-10R1 complex is formed by the association of two 1:2 IL-10/sIL-10R1 complexes. Within the 1:2 IL-10/sIL-10R1 complex each of the helical domains of the IL-10 dimer makes identical interactions with a sIL-10R1 molecule. The conservation of IL-10R1 residues important to this interface in IL-10R2, suggested that IL-10R2 is incorporated into the active complex by making similar interactions with IL-10. Based on this hypothesis and the architecture of the 2:4 IL-10/sIL-10R1 complex we proposed a model for the complex formed between IL-10, IL-10R1, and IL-10R2, and a mechanism for the activation of intracellular signaling.; By engineering a monomeric isomer of 1L-10 and examining its ability to signal, we determined that the IL-10 dimer is not required for signaling, which is consistent with our model of the active IL-10 receptor complex. In order to verify the structural success of our IL-10 engineering we crystallized the IL-10 monomer in complex with the Fab fragment from the neutralizing anti-IL-10 monoclonal antibody 9D7Fab. Surprisingly, the 9D7Fab epitope does not overlap with the IL-10R1 binding site, indicating that 9D7Fab does not competitively antagonize the IL-10/IL-10R1 interaction. By comparing the conformation of the 9D7Fab-bound-IL-10-monomer to IL-10R1-bound-IL-10 we determined that 9D7Fab induces conformational changes in the IL-10R1 binding site that would abrogate the IL-10/IL-10R1 interaction. We confirmed this conclusion by demonstrating that 9D7Fab reduces IL-10R1 affinity ∼250-fold.