Functional implications of large backbone amplitude motions of the glycoprotein 130-binding epitope of interleukin-6.
Human interleukin (IL)-6 plays a pivotal role in the immune response, hematopoiesis, the acute-phase response, and inflammation. IL-6 has three distinct receptor epitopes, termed sites I, II, and III, that facilitate the formation of a signaling complex. IL-6 signals via a homodimer of glycoprotein 130 (gp130) after initially forming a heterodimer with the nonsignaling α-receptor [IL-6 α-receptor (IL-6R)] via site I. Here, we present the backbone dynamics of apo-IL-6 as determined by analysis of NMR relaxation data with the extended model-free formalism of Lipari and Szabo. To alleviate significant resonance overlap in the HSQC-type spectra, cell-free protein synthesis was used to selectively (15) N-label residues, thereby ensuring a complete set of residue-specific dynamics. The calculated order parameters [square of the generalized model-free order parameter (S(2))] showed significant conformational heterogeneity among clusters of residues in IL-6. In particular, the N-terminal region of the long AB-loop, which corresponds spatially to one of the gp130 receptor binding epitopes (i.e. site III), experiences substantial fluctuations along the conformation of the main chain (S(2) = 0.3-0.8) that are not observed at the other two epitopes or in other cytokines. Thus, we postulate that dynamic properties of the AB-loop are responsible for inhibiting the interaction of IL-6 with gp130 in the absence of the IL-6R, and that binding of IL-6R at site I shifts the dynamic equilibrium to favor interaction with gp130 at site III. In addition, molecular dynamics simulations corroborated the NMR-derived dynamics, and showed that the BC-loop adopts different substates that possibly play a role in facilitating receptor assembly.
School of Biological Sciences, University of Auckland, New Zealand; School of Chemical Sciences, University of Auckland, New Zealand.
Romel
R
Bobby
Paul
P
Robustelli
Andrew V
AV
Kralicek
Mehdi
M
Mobli
Glenn F
GF
King
Joachim
J
Grötzinger
Andrew J
AJ
Dingley
D001665:Binding Sites; D050822:Cytokine Receptor gp130 / Q000737:chemistry* / Q000378:metabolism*; D018604:Epitope Mapping; D006801:Humans; D006859:Hydrogen / Q000737:chemistry; D015850:Interleukin-6 / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D009587:Nitrogen Isotopes / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D020836:Protein Structure, Quaternary; D019947:Receptors, Interleukin-6 / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
C508600:IL6 protein, human; C508603:IL6R protein, human; D015850:Interleukin-6; D046912:Multiprotein Complexes; D009587:Nitrogen Isotopes; D019947:Receptors, Interleukin-6; D011994:Recombinant Proteins; D050822:Cytokine Receptor gp130; D006859:Hydrogen
cell-free protein synthesis; cytokine signaling complex; interleukin-6; molecular dynamics simulations; protein backbone dynamics