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Query: UNIPROT:P05231 (interleukin-6)
 23,907 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effects of solvent, pH and temperature on the 1H-NMR spectra of recombinant murine interleukin-6 (IL-6) are described. Assignments made from two-dimensional homonuclear spectra are presented for resonances of the fifteen aromatic amino-acid side chains. A time-dependent loss of intensity was observed for all resonances in the spectrum of IL-6, probably as a result of aggregation. This aggregation is markedly temperature-dependent. The pKa values of the four histidine residues in murine IL-6 has been measured; one has a value of 5.5, approx. one pH unit less than the value exhibited by the other three. Analysis of the NOESY spectra has allowed a preliminary characterisation of the nature of interactions among the aromatic side chains within the protein fold. 1H and 15N resonances of residues Thr-4 to Val-21 are assigned from three-dimensional 1H-15N correlated spectroscopy, and evidence is presented for these residues comprising a mobile N-terminal tail with little ordered structure. An N-terminal mutant lacking the first 22 residues of the murine IL-6 sequence and known to possess full biological activity was also examined and shown to have essentially retained the tertiary fold of the native molecule.
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PMID:NMR studies of the solution properties of recombinant murine interleukin-6. 759 73

A synthetic gene for human interleukin-6 has been expressed in E. coli. The protein has been purified and renatured and has the same activity as natural human IL-6 using the 7TD1 cell proliferation assay. The protein undergoes specific cleavage by a thiol protease, yielding two new N-termini at Arg-9 and His-15. The truncated proteins retain full biological activity. The degradation results in the loss of sharp amide resonances in the 1H-NMR spectrum, and little change to the ultraviolet CD spectrum. Several amino acid type assignments could be made for these sharp amides using a DQF-COSY 2D-NMR experiment. The N-terminal 15 amino acids exist as a flexible, random coil, attached to a central structure.
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PMID:Recombinant human IL-6 expressed in E. coli undergoes selective N-terminal degradation: evidence that the protein consists of a stable core and a nonessential flexible N-terminal. 825 Oct 70

Two synthetic peptides corresponding to the C-terminal 19 residues of human and murine interleukin-6, respectively, have been synthesized and their structures in solution investigated using high-resolution 1H-NMR spectroscopy. Both peptides show a marked dependence of chemical-shift dispersion on pH, with a greater degree of structure apparent above pH 4.5, where their glutamate carboxyl groups are ionised. In purely aqueous solution, neither peptide adopts a well-defined structure, although the murine peptide has characteristics of a nascent helix. Titration of the murine peptide with trifluoroethanol produced a significant increase in structure, which was then investigated using two-dimensional NMR. In 50% (by vol.) trifluoroethanol the murine peptide consists of a well-defined central helix of 12 residues with unstructured N-terminal and C-terminal regions. These observations lend experimental support to the current model of the interleukin-6 structure, which proposes a four-helical bundle with the last helix encompassing the C-terminal 20-30 residues. Furthermore, the fact that synthetic peptides corresponding to part of the putative receptor-binding surface of interleukin-6 are able to adopt a similar conformation in solution to that proposed for the intact protein suggests that such peptide analogues should be useful starting points in the design of peptide agonists and antagonists of interleukin-6.
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PMID:Solution structure of synthetic peptides corresponding to the C-terminal helix of interleukin-6. 830 40

Two murine interleukin-6 (mIL-6) variants were constructed using the polymerase chain reaction (PCR), one lacking the last five residues (183-187) at the C-terminus (pMC5) and another with the last five residues of mIL-6 substituted by the corresponding residues of human IL-6 (pMC5H). The growth stimulatory activity of pMC5 on the mouse hybridoma cell line 7TD1 was < 0.05% of mIL-6, whereas pMC5H and mIL-6 were equipotent. The loss of biological activity of pMC5 correlated with its negligible receptor binding affinity on 7TD1 cells, while the binding of pMC5H was comparable to that of mIL-6. Both pMC5 and pMC5H, like mIL-6, failed to interact with recombinant soluble human IL-6 receptor when assayed by surface plasmon resonance-based biosensor analysis. These studies suggest that the C-terminal seven amino acids of human IL-6, alone, do not define species specificity for receptor binding. A variety of biophysical techniques, as well as the binding of a conformational-specific monoclonal antibody, indicated that the global fold of the mIL-6 variants was similar to that of mIL-6, although small changes in the NMR spectra, particularly for pMC5, were observed. Some of these changes involved residues widely separated in the primary structure. For instance, interactions involving Tyr-22 were influenced by the C-terminal amino acids suggesting that the N- and C-termini of mIL-6 are in close proximity. Equilibrium unfolding experiments indicated that pMC5 was 0.8 kcal/mol less stable than mIL-6, whereas pMC5H was 1.4 kcal/mol more stable. These studies emphasize the structural importance of the C-terminal amino acids of IL-6 and suggest that truncation or mutation of this region could lead to small but significant alterations in other regions of the molecule.
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PMID:Role of the C-terminus in the activity, conformation, and stability of interleukin-6. 840 Dec 31

To understand the structure-function relationship in the human interleukin-6 (IL-6) system, comparative studies were performed on the basis of NMR data obtained using the wild-type IL-6 and six mutants. In each of the six mutants, either Leu152, Leu159, Leu166, Leu168, Leu175, or Leu182, which exist in the C-terminal receptor-binding region, was substituted with Val. The resonance assignments of Val, Ile, Leu, and Phe residues were made by using specific double-labeling and site-specific mutagenesis strategies. On the basis of chemical shift and NOE data collected for six IL-6 mutants and those for the wild-type IL-6, we analyzed the structural changes induced by the substitution of each of the six Leu residues. The NMR data showed that substitution of Leu182 with Val (L182V) induced no structural change in IL-6, suggesting that Leu182 is located on the surface of the IL-6 molecule. A significant decrease in receptor-binding activity was observed in the L182V mutant. It was concluded that the side chain of Leu182 is directly involved in receptor binding. Substitution of Leu175 with Val (L175V) was shown to induce a significant structural change in IL-6. The NMR data are discussed on the basis of the location of four helix elements and an up-up-down-down helix topology of the predicted structure of IL-6 [Bazan, J.F. (1991) Neuron 7, 197-208]. It is possible that helix D bent more sharply toward helix B in the L175V mutant than in the wild-type IL-6 to maintain a closely packed and solvent-inaccessible core formed in the mutated region. It is suggested that the kink of helix D is related to the decrease in receptor-binding activity in the L175V mutant. On the basis of the observed NOE network, the folding topology of IL-6 was analyzed. A comparison of the folding topology of IL-6 with that of human granulocyte colony-stimulating factor determined by X-ray crystallography [Hill, C. P., Osslund, T. D., & Eisenberg, D. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 5167-5171] indicated that IL-6 has a significant similarity of folding topology to that of human granulocyte colony-stimulating factor.
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PMID:Folding topologies of human interleukin-6 and its mutants as studied by NMR spectroscopy. 855 85

Oncostatin M (OM) is a cytokine that shares a structural and functional relationship with interleukin-6, leukemia inhibitory factor, and granulocyte-colony stimulating factor, which regulate the proliferation and differentiation of a variety of cell types. A mutant version of human OM in which two N-linked glycosylation sites and an unpaired cysteine have been mutated to alanine (N76A/C81A/N193A) has been expressed and shown to be active. The triple mutant has been doubly isotope-labeled with 13C and 15N in order to utilize heteronuclear multidimensional NMR techniques for structure determination. Approximately 90% of the backbone resonances were assigned from a combination of triple-resonance data (HNCA, HNCO, CBCACONH, HBHACONH, HNHA and HCACO), intraresidue and sequential NOEs (3D 15N-NOESY-HMQC and 13C-HSQC-NOESY) and side-chain information obtained from the CCONH and HCCONH experiments. Preliminary analysis of the NOE pattern in the 15N-NOESY-HMQC spectrum and the 13C alpha secondary chemical shifts predicts a secondary structure for OM consisting of four alpha-helices with three intervening helical regions, consistent with the four-helix-bundle motif found for this cytokine family. As a 203-residue protein with a molecular weight of 24 kDa, Oncostatin M is the largest alpha-helical protein yet assigned.
J Biomol NMR 1996 Jun
PMID:Resonance assignments for Oncostatin M, a 24-kDa alpha-helical protein. 876 35

Essentially complete backbone and side-chain 1H, 15N and 13C resonance assignments for the 185-amino-acid cytokine interleukin-6 (IL-6) are presented. NMR experiments were performed on uniformly [15N]- and [15N,13C]-labeled recombinant human IL-6 (rIL-6) using a variety of heteronuclear NMR experiments. A combination of 13C-chemical shift, amide hydrogen-bond exchange, and 15N-edited NOESY data allowed for analysis of the secondary structure of IL-6. The observed secondary structure of IL-6 is composed of loop regions connecting five alpha-helices, four of which are consistent in their length and disposition with the four-helix bundle motif present in other related cytokines and previously postulated for IL-6. In addition, the topology of the overall fold was found to be consistent with a left-handed up-up-down-down four-helix bundle based on a number of long-range interhelical NOEs. The results presented here provide deeper insight into structure-function relationships among members of the four-helix bundle family of proteins.
J Biomol NMR 1996 Sep
PMID:Complete 1H, 15N and 13C assignments, secondary structure, and topology of recombinant human interleukin-6. 891 71

Tertiary structure models of Interleukin-6 were constructed using a routine prediction method based on the X-ray crystal structures of granulocyte colony-stimulating factor (GCSF) and leukemia inhibitory factor (LIF). The models were evaluated with the aid of the sequence-structure compatibility (3D-1D) method program compass and NMR experimental information. The model constructed from GCSF gained higher scores on compass examination than did that from LIF, and the NOE data [Nishimura et al. (1996) Biochemistry 35, 273-281] also turned to be more consistent with the former model.
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PMID:Tertiary structural models for human interleukin-6 and evaluation by a sequence-structure compatibility method and NMR experimental information. 911 70

Interleukin-6 (IL-6) is a 185 amino acid cytokine which exerts multiple biological effects in vivo and whose dysregulation underlies several disease processes. The solution structure of recombinant human interleukin-6 has now been determined using heteronuclear three and four-dimensional NMR spectroscopy. The structure of the molecule was determined using 3044 distance and torsion restraints derived by NMR spectroscopy to generate an ensemble of 32 structures using a combined distance geometry/simulated annealing protocol. The protein contains five alpha-helices interspersed with variable-length loops; four of these helices constitute a classical four-helix bundle with the fifth helix located in the CD loop. There were no distance violations greater than 0.3 A in any of the final 32 structures and the ensemble has an average-to-the-mean backbone root-mean-square deviation of 0.50 A for the core four-helix bundle. Although the amino-terminal 19 amino acids are disordered in solution, the remainder of the molecule has a well defined structure that shares many features displayed by other long-chain four-helix bundle cytokines. The high-resolution NMR structure of hIL-6 is used to rationalize available mutagenesis data in terms of a heteromeric receptor complex.
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PMID:Solution structure of recombinant human interleukin-6. 915 84

Interleukin-6 (IL-6) is a multifunctional cytokine which is involved in a broad spectrum of activities such as immune defense, hematopoiesis, and the acute phase response, as well as in the pathogenesis of multiple myeloma. A series of murine IL-6 (mIL-6) mutants, H31A, W34A, and H31A/W34A, were constructed to investigate the roles of His31 and Trp34 in the structure, conformational stability, time-dependent aggregation, folding, and spectral properties of mIL-6. The characteristic pH-dependent quenching of fluorescence of mIL-6 at low pH was shown to be caused by an interaction between Trp34 and protonated His31 at low pH and not associated with Trp157. Denaturant-induced equilibrium unfolding experiments monitored by fluorescence and far-UV CD showed that the increased quantum yield and blue shift of the wavelength of the emission maximum observed for mIL-6 at moderate denaturant concentrations were also associated with Trp34, rather than Trp157. The tendency to form aggregation-prone unfolding intermediates, as judged by poor fits to a two-state unfolding mechanism, low m values (slopes of the unfolding curve in the transition region), and the range of denaturant concentrations over which these intermediates formed, was shown to be higher for H31A than mIL-6 but significantly lower for W34A and H31A/W34A. These differences were most pronounced at pH 7.4 and correlated with the tendencies of the proteins to aggregate at high protein concentrations in the absence of denaturant. As judged by the 1H NMR chemical shifts of the aromatic residues, the global conformations of H31A and W34A were not significantly different from that of mIL-6. Nuclear Overhauser effects (NOE) between the side chains of His31 and Trp34 were consistent with the indole side chain of Trp34 being oriented toward the face of the imidazolium side chain of His31, an arrangement consistent with our estimates of a low interaction energy (0.4-0.6 kcal/mol) between these side chains. A shift in the pKa of the His31 side chain in W34A (+0.3 unit) suggested that, in the absence of Trp34, His31 could interact with other residues. Further mutations in this region should yield forms of mIL-6, even less prone to aggregation, which would be more suitable for NMR studies. Mutation of His31 and Trp34 to alanine did not significantly alter the mitogenic activity of the mutants on mouse hybridoma 7TD1 cells, even though the corresponding region of human IL-6 has been shown to be important for biological activity.
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PMID:Roles of histidine 31 and tryptophan 34 in the structure, self-association, and folding of murine interleukin-6. 916 91


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