Characterization of an antagonist interleukin-6 dimer by stable isotope labeling, cross-linking, and mass spectrometry

The homodimeric form of a recombinant cytokine interleukin-6 (IL-6(D)) is known to antagonize IL-6 signaling. In this study, spatially proximal residues between IL-6 chains in IL-6, were identified using a method for specific recognition of intermolecular cross-linked peptides. Our strategy involved mixing 1:1 N-15-labeled and unlabeled (N-14) protein to form a mixture of isotopically labeled and unlabeled homodimers, which was chemically cross-linked. This cross-linked IL-6(D) was subjected to proteolysis by trypsin and the generated peptides were analyzed by electrospray ionization time-of-flight mass spectrometry (MS). Molecular ions from crosslinked peptides of intermolecular origin are labeled with [N-15/N-15] + [N-15/N-14] + [N-14/N-15] + [N-14/N-14] yielding readily identified triplet/quadruplet MS peaks. All other peptide species are labeled with [N-15] + [N-14] yielding doublet peaks. Intermolecular cross-linked peptides were identified by MS, and cross-linked residues were identified. This intermolecular cross-link detection method, which we have designated "mixed isotope cross-linking" MIX may have more general application to protein-protein interaction studies. The pattern of proximal residues found was consistent with IL-6(D) having a domain-swapped fold similar to IL-10 and interferon-gamma. This fold implies that IL-6(D)-mediated antagonism of IL-6 signaling is caused by obstruction of cooperative gp130 binding on IL-6(D), rather than direct blocking of gp-130-binding sites on IL-6(D).