Roles of disulfide bonds in recombinant human interleukin 6 conformation.


Abstract

Human IL-6 has two disulfide bonds linking Cys45 to Cys51 and Cys74 to Cys84, respectively. Previous site-directed mutagenesis studies have demonstrated that the Cys74-Cys84 bond is essential for full biological and receptor binding activities. To address the structural importance of these disulfide bonds in the formation and stabilization of IL-6 secondary and tertiary structures, we have generated a panel of disulfide bond-deficient rIL-6 analogs both by chemical reduction and alkylation as well as by site-directed mutagenesis. Conformational changes affecting these rIL-6 analogs were probed by circular dichroism spectroscopy, as well as reactivity with monoclonal antibodies, and correlated with changes in biological activities. We have shown that the first disulfide bridge (Cys45-Cys51) is highly sensitive to reduction and, therefore, more solvent-exposed or less thermodynamically stable. Contrary to previous reports, this bridge contributes, although minimally, to the full biological activity of the cytokine. However, no significant changes in secondary or tertiary structures were observed upon removal of this bond. In marked contrast, analogs lacking the disulfide bridge between Cys74 and Cys84 exhibited as little as 0.5% and 0.05% wild-type biological and receptor binding activities, respectively. These dramatic changes correlated with a slight reduction in alpha-helical content and a decreased reactivity with the neutralizing monoclonal antibody mAb8 which recognizes a conformational epitope associated with the active site. Our results suggest that the second disulfide bridge plays a critical role in maintaining the spatial relationship between the putative IL-6 A and D helices. Study holds ProTherm entries: 4443 Extra Details: disulfide bonds; receptor binding activities;,secondary and tertiary structures; conformational changes

Submission Details

ID: fxehifz6

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:25 p.m.

Version: 1

Publication Details
Rock FL;Li X;Chong P;Ida N;Klein M,Biochemistry (1994) Roles of disulfide bonds in recombinant human interleukin 6 conformation. PMID:8172889
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
2IL6 1998-02-04 HUMAN INTERLEUKIN-6, NMR, 32 STRUCTURES
1IL6 1998-02-04 HUMAN INTERLEUKIN-6, NMR, MINIMIZED AVERAGE STRUCTURE
1ALU 1998-06-03 1.9 HUMAN INTERLEUKIN-6
4CNI 2014-04-30 2.2 Crystal structure of the Fab portion of Olokizumab in complex with IL- 6
4J4L 2014-02-12 2.3 Modular evolution and design of the protein binding interface
4NI7 2014-01-22 2.4 Crystal structure of human interleukin 6 in complex with a modified nucleotide aptamer (SOMAMER SL1025)
4O9H 2015-04-15 2.42 Structure of Interleukin-6 in complex with a Camelid Fab fragment
4NI9 2014-01-22 2.55 Crystal structure of human interleukin 6 in complex with a modified nucleotide aptamer (SOMAMER SL1025), FORM 2
5FUC 2017-01-25 2.7 Biophysical and cellular characterisation of a junctional epitope antibody that locks IL-6 and gp80 together in a stable complex: implications for new therapeutic strategies
4ZS7 2016-05-04 2.93 Structural mimicry of receptor interaction by antagonistic IL-6 antibodies
1P9M 2003-07-01 3.65 Crystal structure of the hexameric human IL-6/IL-6 alpha receptor/gp130 complex

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
95.8 Interleukin-6 P46650 IL6_CERAT
96.2 Interleukin-6 Q5I6E3 IL6_MACTH
96.2 Interleukin-6 P51494 IL6_MACMU
96.7 Interleukin-6 P79341 IL6_MACFA
100.0 Interleukin-6 P05231 IL6_HUMAN