Human interleukin 4: The solution structure of a four-helix bundle protein

doi:10.1016/0022-2836(92)90457-U

Journal of Molecular Biology

Volume 224, Issue 4, 20 April 1992, Pages 899-904

https://doi.org/10.1016/0022-2836(92)90457-U Get rights and content

Abstract

Heteronuclear ¹³C and ¹⁵N three-dimensional nuclear magnetic resonance (n.m.r.) techniques have been used to determine the solution structure of human interleukin 4, a four-helix bundle protein. A dynamical simulated annealing protocol was used to calculate an ensemble of structures from an n.m.r. data set of 1735 distance restraints, 101 φ angle restraints and 27 pairs of hydrogen bond restraints. The protein structure has a left-handed up-up-down-down topology for the four helices with the two long overhand loops in the structure being connected by a short section of irregular antiparallel β-sheet. Analysis of the side-chains in the protein shows a clustering of hydrophobic residues, particularly leucines, in the core of the bundle with the side-chains of charged residues being located on the protein surface. The solution structure has been compared with a recent structure prediction for human interleukin 4 and with crystal structures of other helix bundle proteins.

References (24)

A. Bax et al.
¹H-¹H correlation via isotropic mixing of ¹³C magnetization, a new three-dimensional approach for assigning ¹H and ¹³C spectra of ¹³C-enriched proteins
J. Magn. Reson
(1990)
J.F. Bazan
Haemopoietic receptors and helical cytokines
Immunol. Today
(1990)
L.E. Kay et al.
Three-dimensional triple-resonance n.m.r. spectroscopy of isotopically enriched proteins
J. Magn. Reson
(1990)
N. Kruse et al.
Site-directed mutagenesis reveals the importance of disulfide bridges and aromatic residues for structure and proliferative activity of human interleukin-4
FEBS Letters
(1991)
M. Nilges et al.
Determination of three-dimensional structures of proteins from interproton distance data by hybrid distance geometry-dynamical simulated annealing calculations
FEBS Letters
(1988)
L.H. Weaver et al.
Structure of bacteriophage T4 lysozyme refined at 1·7 Å resolution
J. Mol. Biol
(1987)
M.P. Williamson et al.
Solution conformation of proteinase inhibitor IIa from bull seminal plasma by ¹H nuclear magnetic resonance and distance geometry
J. Mol. Biol
(1985)
K. Wüthrich et al.
Pseudo-structures for the 20 common amino acids for use in studies of protein conformations by measurements of intramolecular proton-proton distance constraints with nuclear magnetic resonance
J. Mol. Biol
(1983)
S.S. Abdel-Meguid et al.
Three-dimensional structure of a genetically engineered variant of porcine growth hormone
B.J. Brandhuber et al.
Three-dimensional structure of interleukin-2
Science
(1987)

A.T. Brünger

C. Carr et al.

Disulfide assignments in recombinant mouse and human interleukin 4

Biochemistry

(1991)

Cited by (127)

A specialized method of sputum collection and processing for therapeutic interventions in cystic fibrosis
2019, Journal of Cystic Fibrosis
Citation Excerpt :
Other sputum inflammatory biomarkers that have been associated with clinical events such as exacerbations include calprotectin [74], high-mobility group box 1 (HMGB-1) [75] and YKL-40 [76]. A contributing factor to the variability and suboptimal reproducibility seen in sputum is the acquisition, handling and processing of the samples, which in many cases involves the use of the reducing agent dithiothreitol (DTT) [70, 73, 77–92]. In addition, there is also the possibility that sampling only PWCF who spontaneously expectorate sputum may result in a biased, “sicker” population, whereas relying on those PWCF who are fit for BAL may bias the population to a less sick cohort.
Cystic fibrosis (CF) lung disease is characterized by aggressive neutrophil-dominated inflammation mediated in large part by neutrophil elastase (NE), an omnivorous protease released by activated or disintegrating neutrophils and a key therapeutic target. To date, several short-term studies have shown that anti-NE compounds can inhibit NE and have anti-inflammatory effects. However, progression to large-scale or multicenter clinical trials has been hampered by the fact that the current gold standard methodology of evaluating airway NE inhibition, bronchoalveolar lavage (BAL), is invasive, difficult to standardize across sites and excludes those with severe lung disease. Attempts to utilize sputum that is either spontaneously expectorated (SS) or induced (IS) have been hindered by poor reproducibility, often due to the various processing methods employed. In this study, we evaluate TEmperature-controlled Two-step Rapid Isolation of Sputum (TETRIS), a specialized method for the acquisition and processing of SS and IS. Using TETRIS, we show for the first time that NE activity and cytokine levels are comparable in BAL, SS and IS samples taken from the same people with CF (PWCF) on the same day once this protocol is used. We correlate biomarkers in TETRIS-processed IS and clinical outcome measures including FEV₁, and show stability and reproducible inhibition of NE over time in IS processed by TETRIS. The data offer a tremendous opportunity to evaluate prognosis and therapeutic interventions in CF and to study the full spectrum of people with PWCF, many of whom have been excluded from previous studies due to being unfit for BAL or unable to expectorate sputum.
Characterization of cryptic allosteric site at IL-4Rα: New paradigm towards IL-4/IL-4R inhibition
2019, International Journal of Biological Macromolecules
Citation Excerpt :
So far, only the extracellular binding region of IL-4Ra has been resolved, comprising of two fibronectin-III (fnIII) type domains each having stretch of 100 residues which are connected by a linker region [10]. While the ligand protein (IL-4) binding with receptor is a four-helix complex [11,12] arranged in anti-parallel manner with two elongated loops linked by a short beta-sheet structure [13–18]. The charge complementarity between the crucial residues of IL-4 and its receptor alpha plays a key role in their protein-protein interactions [19].
Interleukin-4(IL-4), an anti-inflammatory cytokine, plays significant role in pathogenesis of various diseases such as asthma, tumors, and HIV infections. These responses are mediated by expression of IL-4R (receptor) on various hematopoietic and non-hematopoietic cells surfaces. To date, the X-ray crystal structure of unbound (i.e. free) IL-4R is not reported which hampers active research on the molecular interaction mechanism between IL-4 and IL-4R. To investigate the missing gaps about stable binding mode of IL-4 and drug-ability of IL-4R active site, modelling and molecular dynamics (MD) simulation of IL-4/IL-4R complex was performed. Drug-ability of the target protein changed after modelling the loop region near C-terminal of IL-4R protein. This led to the identification of a novel druggable site other than the reported interfacial site. Our analysis showed that the modelled residues Ser111 and Ser164-Lys167 are part of newly discovered allosteric site, which underwent major fluctuation after association with its ligand protein (IL-4). The results indicated possible role of this cryptic allosteric site in IL-4/IL-4R signaling pathway that might help us to block IL-4/IL-4R association to prevent various allergic and malignant diseases.
Interleukin-4 receptor signaling and its binding mechanism: A therapeutic insight from inhibitors tool box
2016, Cytokine and Growth Factor Reviews
Citation Excerpt :
SHP-1 tyrosine phosphorylation enhances its phosphatase agility [64],while SHP-2 phosphorylation generates binding site for proteins having SH2 subunits such as IRS-1, p85, Grb2 [65]. Irina G. Luzina et al. verified that SHP-1 reduces STAT-6 signaling by hydrolyzing phosphatidylinositol (3–5) triphosphate and the product of PI3 kinase activity via type-I receptor complex [17,66]. Reduction in IL-4/IL-13 signaling is also achieved by accelerated activation of SOCS (suppressor of cytokine signaling) proteins [67].
Studies on Interlukin-4 (IL-4) disclosed great deal of information about its various physiological and pathological roles. All these roles depend upon its interaction and signaling through either type-I (IL-4Rα/common γ-chain) or type-II (IL-4Rα/IL-13Rα) receptors. Another cytokine, IL-13, shares some of the functions of IL-4, because both cytokines use a common receptor subunit, IL-4Rα. Here in this review, we discuss the structural details of IL-4 and IL-4Rα subunit and the structural similarities between IL-4 and IL-13. We also describe detailed chemistry of type-I and type-II receptor complexes and their signaling pathways. Furthermore, we elaborate the strength of type-II hetero dimer signals in response to IL-4 and IL-13. These cytokines are prime players in pathogenesis of allergic asthma, allergic hypersensitivity, different cancers, and HIV infection. Recent advances in the structural and binding chemistry of these cytokines various types of inhibitors were designed to block the interaction of IL-4 and IL-13 with their receptor, including several IL-4 mutant analogs and IL-4 antagonistic antibodies. Moreover, different targeted immunotoxins, which is a fusion of cytokine protein with a toxin or suicidal gene, are the new class of inhibitors to prevent cancer progression. In addition few small molecular inhibitors such as flavonoids have also been developed which are capable of binding with high affinity to IL-4Rα and, therefore, can be very effective in blocking IL-4-mediated responses.
The existence of multiple conformers of interleukin-21 directs engineering of a superpotent analogue
2007, Journal of Biological Chemistry
Citation Excerpt :
A common feature for hIL-2, hIL-4, and hIL-21 is the disordered CD loop. NMR structures have previously been reported for IL-2 (4) and IL-4 (5, 6, 25). Compared with the four-helix bundle, the CD loop, excluding the β-sheet, is poorly defined in both structures.
The high resolution three-dimensional structure of human interleukin (hIL)-21 has been resolved by heteronuclear NMR spectroscopy. Overall, the hIL-21 structure is dominated by a well defined central four-helical bundle, arranged in an up-up-down-down topology, as observed for other cytokines. A segment of the hIL-21 molecule that includes the third helical segment, helix C, is observed to exist in two distinct and interchangeable states. In one conformer, the helix C segment is presented in a regular, α-helical conformation, whereas in the other conformer, this segment is largely disordered. A structure-based sequence alignment of hIL-21 with receptor complexes of the related cytokines, interleukin-2 and -4, implied that this particular segment is involved in receptor binding. An hIL-21 analog was designed to stabilize the region around helix C through the introduction of a segment grafted from hIL-4. This novel hIL-21 analog was demonstrated to exhibit a 10-fold increase in potency in a cellular assay.
On-column refolding of recombinant human interleukin-4 from inclusion bodies
2004, Protein Expression and Purification
Interleukin-4 (IL4) is a multifunctional cytokine which plays a key role in the immune system. Several antagonists/agonists of IL4 are reported through mutagenesis studies, but their solution structural studies using nuclear magnetic resonance (NMR) spectroscopy are hindered as milligram quantities of isotopically labeled protein are required for structural refinements. In this work, a His-tagged recombinant form of human IL4 was overexpressed in Escherichia coli under the control of a T7 promoter. The resulting inclusion bodies were separated from cellular debris by centrifugation and solubilized by 6 M guanidine–HCl in the presence of reducing agents. The denatured IL4 was immobilized on Ni²⁺-fractogel beads and refolded in a single chromatographic step by gradual removal of denaturant. This protocol yielded 15–20 mg of isotope-enriched protein from 1 L of culture grown in minimal medium. The refolded protein was highly pure and was correctly folded as judged by its two-dimensional NMR spectrum. To show the successful application of this refolding protocol to IL4 variants, ¹⁵N-labeled Y124D-IL4 was also prepared and its first two-dimensional NMR spectrum was presented.
Compensatory energetic mechanisms mediating the assembly of signaling complexes between interleukin-2 and its α, β, and γ<inf>c</inf> receptors
2004, Journal of Molecular Biology
Interleukin-2 is a key immuno-regulatory cytokine whose actions are mediated by three different cell surface receptors: the α, β and the “common γ” (γ_c) chains. We have undertaken a complete thermodynamic characterization of the stepwise assembly cycle for multiple possible combinations of the receptor–ligand, and receptor–receptor interactions that are necessary for formation of the high-affinity IL-2/αβγ_c signaling complex. We find an entropically favorable high affinity interaction between IL-2 and its α receptor, a moderately entropically favorable low affinity interaction between IL-2 and its β receptor, and no interaction between IL-2 and the shared receptor, γ_c. Formation of the stable intermediate trimolecular complexes of IL-2 with α and β receptors, as well as IL-2 with β and γ_c receptors proceeds through enthalpy–entropy compensation mechanisms. Surprisingly, we see a moderate affinity interaction between the unliganded receptor α and β chains, suggesting that a preformed αβ complex may serve as the initial interaction complex for IL-2. Reconstitution of the IL-2/Rαβγ_c high-affinity quaternary signaling complex shows it to be assembled through cooperative energetics to form a 1:1:1:1 assembly. Collectively, the favorable entropy of the bimolecular interactions appears to be offset by the loss in rigid body entropy of the receptor components in the higher-order complexes, but overcome by the formation of increasingly enthalpically favorable composite interfaces. This enthalpic mechanism utilized by γ_c contrasts with the favorable entropic mechanism utilized by gp130 for degenerate cytokine interaction. In conclusion, we find that several energetically redundant pathways exist for formation of IL-2 receptor signaling complexes, suggesting a more complex equilibrium on the cell surface than has been previously appreciated.

View all citing articles on Scopus

^☆: This is a contribution from the Oxford Centre for Molecular Sciences which is supported by the U.K. Science and Engineering Research Council and the Medical Research Council.

View full text

Article preview

Journal of Molecular Biology

Abstract

References (24)

¹H-¹H correlation via isotropic mixing of ¹³C magnetization, a new three-dimensional approach for assigning ¹H and ¹³C spectra of ¹³C-enriched proteins

J. Magn. Reson

Haemopoietic receptors and helical cytokines

Immunol. Today

Three-dimensional triple-resonance n.m.r. spectroscopy of isotopically enriched proteins

J. Magn. Reson

Site-directed mutagenesis reveals the importance of disulfide bridges and aromatic residues for structure and proliferative activity of human interleukin-4

FEBS Letters

Determination of three-dimensional structures of proteins from interproton distance data by hybrid distance geometry-dynamical simulated annealing calculations

FEBS Letters

Structure of bacteriophage T4 lysozyme refined at 1·7 Å resolution

J. Mol. Biol

Solution conformation of proteinase inhibitor IIa from bull seminal plasma by ¹H nuclear magnetic resonance and distance geometry

J. Mol. Biol

Pseudo-structures for the 20 common amino acids for use in studies of protein conformations by measurements of intramolecular proton-proton distance constraints with nuclear magnetic resonance

J. Mol. Biol

Three-dimensional structure of a genetically engineered variant of porcine growth hormone

Three-dimensional structure of interleukin-2

Science

Disulfide assignments in recombinant mouse and human interleukin 4

Biochemistry

Cited by (127)

A specialized method of sputum collection and processing for therapeutic interventions in cystic fibrosis

Characterization of cryptic allosteric site at IL-4Rα: New paradigm towards IL-4/IL-4R inhibition

Interleukin-4 receptor signaling and its binding mechanism: A therapeutic insight from inhibitors tool box

The existence of multiple conformers of interleukin-21 directs engineering of a superpotent analogue

On-column refolding of recombinant human interleukin-4 from inclusion bodies

Compensatory energetic mechanisms mediating the assembly of signaling complexes between interleukin-2 and its α, β, and γ<inf>c</inf> receptors

Journal of Molecular Biology

CommunicationHuman interleukin 4: The solution structure of a four-helix bundle protein☆

Abstract

J. Magn. Reson

Immunol. Today

J. Magn. Reson

FEBS Letters

FEBS Letters

J. Mol. Biol

J. Mol. Biol

J. Mol. Biol

Three-dimensional structure of a genetically engineered variant of porcine growth hormone

Three-dimensional structure of interleukin-2

Science

Disulfide assignments in recombinant mouse and human interleukin 4

Biochemistry

Communication
Human interleukin 4: The solution structure of a four-helix bundle protein☆