Structure, stability and biological properties of a N-terminally truncated form of recombinant human interleukin-6 containing a single disulfide bond.


Abstract

A mutant species of the 185-residue chain of human interleukin-6 lacking 22-residues at its N-terminus and with a Cys-->Ser substitution at positions 45 and 51 was produced in Escherichia coli. The 163-residue protein des-(A1-S22)-[C45S, C51S]interleukin-6, containing a single disulfide bridge, formed inclusion bodies. Mutant interleukin-6 was solubilized in 6 M guanidine hydrochloride, subjected to oxidative refolding and purified to homogeneity by ammonium sulfate precipitation and hydrophobic chromatography. The purity of the mutant species was established by electrophoresis, isoelectrofocusing and reverse-phase HPLC and its structural identity was checked by N-terminal sequencing of both the intact protein and several of its proteolytic fragments. Electrospray mass spectrometry analysis of mutant interleukin-6 gave a molecular mass of 18,695 +/- 2 Da in excellent agreement with the calculated value. Circular dichroic, fluorescence emission and second-derivative ultraviolet absorption spectra indicated that mutant interleukin-6 maintains the overall secondary and tertiary structure, as well as stability characteristics, of the recombinant wild-type human interleukin-6. The urea-induced unfolding of mutant interleukin-6, monitored by circular dichroic measurements in the far-ultraviolet region, occurs as a highly cooperative process with a midpoint of denaturation at 5.5 M urea. The data of the reversible unfolding of mutant interleukin-6 mediated by urea were used to calculate a value of 20.9 +/- 0.4 kJ.mol-1 for the thermodynamic stability of the protein at 25 degrees C in the absence of denaturant. The biological activity of mutant interleukin-6 was evaluated in vitro by the hybridoma proliferation assay, and in vivo by measuring thrombopoiesis in monkeys. Dose/response effects of the mutant were comparable or even higher than those of the wild-type protein. Overall the results of this study show that mutant interleukin-6 is a biologically active cytokine, which could find practical use as a therapeutic agent. Study holds ProTherm entries: 8730 Extra Details: interleukin-6; protein engineering; disulfide bond substitution;,protein stability; circular dichroism

Submission Details

ID: cJXFQXZ93

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:36 p.m.

Version: 1

Publication Details
Breton J;La Fiura A;Bertolero F;Orsini G;Valsasina B;Ziliotto R;De Filippis V;Polverino de Laureto P;Fontana A,Eur. J. Biochem. (1995) Structure, stability and biological properties of a N-terminally truncated form of recombinant human interleukin-6 containing a single disulfide bond. PMID:7851440
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
1IL6 1998-02-04 HUMAN INTERLEUKIN-6, NMR, MINIMIZED AVERAGE STRUCTURE
2IL6 1998-02-04 HUMAN INTERLEUKIN-6, NMR, 32 STRUCTURES
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