Conservation of Functional Sites on Interleukin-6 and Implications for Evolution of Signaling Complex Assembly and Therapeutic Intervention

Abstract

Background: Interleukin-6 signaling requires assembly of a ternary IL-6/IL-6R␣/gp130 complex. Results: Determination of the mIL-6 structure allowed detailed structural and sequence comparisons with hIL-6, predicting the primacy of sites in driving IL-6/IL-6R␣-gp130 interactions, which was confirmed by binding experiments. Conclusion:Interactions between gp130 domain-1 and IL-6/IL-6R␣ drive signaling complex assembly. Significance: This suggests a pathway for evolution of signaling complex assembly and strategies for therapeutic targeting.

Novel Insights into Interleukin 6 (IL-6) Cis- and Trans-signaling Pathways by Differentially Manipulating the Assembly of the IL-6 Signaling Complex

“…3C). Taken together, our results show that the binding of 25F10 to mIL-6R requires the glutamic acid at position 261 in D3, which is a residue that has been described to be key in the interaction site IIb between IL-6R (i) and gp130 (i) for the formation of the trimeric IL-6⅐IL-6R⅐gp130 complex (17).…”

Conformational Heterogeneity in Antibody-Protein Antigen Recognition

“…To produce the anti-IL-6 scFv, the parent Fab vector was modified by restriction digestion and PCR to produce a single expressible gene encoding the variable light domain (V L ), (Gly 4 Ser) 4 linker, variable heavy domain (V H ) and His 6 tag, as described previously for an anti-IL-1␤ scFv (14). pET-21-based vectors encoding IL-1␤ or IL-6 with an N-terminal His 6 tag containing a tobacco etch virus protease site in the linker were also provided by UCB (14,15 (16,17) and expression induced at 37°C (14). Uniformly 15 N labeled and unlabeled anti-IL-6 scFv were similarly expressed; however, 15 N/ 2 H-and 15 N/ 13 C/ 2 H-labeled proteins were produced using a small volume, high cell density expression procedure (18) to obtain high yields of deuterated protein from 50-ml cultures.…”

“…pET-21-based vectors encoding IL-1␤ or IL-6 with an N-terminal His 6 tag containing a tobacco etch virus protease site in the linker were also provided by UCB (14,15 (16,17) and expression induced at 37°C (14). Uniformly 15 N labeled and unlabeled anti-IL-6 scFv were similarly expressed; however, 15 N/ 2 H-and 15 N/ 13 C/ 2 H-labeled proteins were produced using a small volume, high cell density expression procedure (18) to obtain high yields of deuterated protein from 50-ml cultures.…”

“…Surface Plasmon Resonance-On and off rate constants for antigen binding to immobilized antibody fragments were determined by surface plasmon resonance experiments carried out essentially as described previously (15).…”

“…In this paper, we report the acquisition and analysis of high quality three-dimensional 15 N/ 1 H and 15 N/ 13 C/ 1 H NMR spectra for both free and antigen-bound scFvs and Fabs. This has allowed the determination of comprehensive sequence-specific backbone resonance assignments for the antibody fragments, which have revealed conserved structural changes within distinct antibody fragments upon binding to structurally diverse protein antigens.…”