Comparative model building of interleukin-7 using interleukin-4 as a template: a structural hypothesis that displays atypical surface chemistry in helix D important for receptor activation

Using a combination of theoretical sequence structure recognition predictions and experimental disulfide bond assignments, a three-dimensional (3D) model of human interleukin-7 (hIL-7) was constructed that predicts atypical surface chemistry in helix D that is important for receptor activation. A 3D model of hIL-7 was built using the X-ray crystal structure of interleukin-4 (IL-4) as a template (Walter MR et al., 1992, J Mol Biol. 224:1075-1085; Walter MR et al., 1992, J Biol Chem 267:20371-20376). Core secondary structures were constructed from sequences of hIL-7 predicted to form helices. The model was constructed by superimposing IL-7 helices onto the IL-4 template and connecting them together in an up-up down-down topology. The model was finished by incorporating the disulfide bond assignments (Cys3, Cys142), (Cys35, Cys130), and (Cys48, Cys93), which were determined by MALDI mass spectroscopy and site-directed mutagenesis (Cosenza L, Sweeney E, Murphy JR, 1997, J Biol Chem 272:32995-33000). Quality analysis of the hIL-7 model identified poor structural features in the carboxyl terminus that, when further studied using hydrophobic moment analysis, detected an atypical structural property in helix D, which contains Cys 130 and Cys142. This analysis demonstrated that helix D had a hydrophobic surface exposed to bulk solvent that accounted for the poor quality of the model, but was suggestive of a region in IL-7 that maybe important for protein interactions. Alanine (Ala) substitution scanning mutagenesis was performed to test if the predicted atypical surface chemistry of helix D in the hIL-7 model is important for receptor activation. This analysis resulted in the construction, purification, and characterization of four hIL-7 variants, hIL-7(K121A), hIL-7(L136A), hIL-7(K140A), and hIL-7(W143A), that displayed reduced or abrogated ability to stimulate a murine IL-7 dependent pre-B cell proliferation. The mutant hIL-7(W143A), which is biologically inactive and displaces [125I]-hIL-7, is the first reported IL-7R system antagonist.     

Structural requirements for gp80 independence of human herpesvirus 8 interleukin-6 (vIL-6) and evidence for gp80 stabilization of gp130 signaling complexes induced by vIL-6

Human herpesvirus 8 interleukin-6 (vIL-6) displays 25% amino acid identity with human IL-6 (hIL-6) and shares an overall four-helix-bundle structure and gp130-mediated STAT/mitogen-activated protein kinase signaling with its cellular counterpart. However, vIL-6 is distinct in that it can signal through gp130 alone, in the absence of the nonsignaling gp80 alpha-subunit of the IL-6 receptor. To investigate the structural requirements for gp80 independence of vIL-6, a series of expression vectors encoding vIL-6/hIL-6 chimeric and site-mutated IL-6 proteins was generated. The replacement of hIL-6 residues with three vIL-6-specific tryptophans implicated in gp80 independence from crystallographic studies or the A and C helices containing these residues did not confer gp80 independence to hIL-6. The N- and C-terminal regions of vIL-6 could be substituted with hIL-6 sequences with the retention of gp80-independent signaling, but substitutions of other regions of vIL-6 (helix A, A/B loop, helix B, helix C, and proximal half of helix D) with equivalent sequences of hIL-6 abolished gp80 independence. Interestingly, the B helix of vIL-6 was absolutely required for gp80 independence, despite the fact that this region contains no receptor-binding residues. Point mutational analysis of helix C, which contains residues involved in physical and functional interactions with gp130 domains 2 and 3 (cytokine-binding homology region), identified a variant, VI120EE, that was able to signal and dimerize gp130 only in the presence of gp80. gp80 was also found to stabilize gp130:g130 dimers induced by a distal D helix variant of vIL-6 that was nonetheless able to signal independently of gp80. Together, our data reveal the crucial importance of overall vIL-6 structure and conformation for gp80-independent signaling and provide functional and physical evidence of the stabilization of vIL-6-induced gp130 signaling complexes by gp80.     

Immunological and structural homology between human T-cell leukemia virus type I envelope glycoprotein and a region of human interleukin-2 implicated in binding the beta receptor.

The N-terminal segment of human interleukin-2 (hIL-2) appears to mediate binding of the beta hIL-2 receptor (R. Robb, C. Rusk, J. Yodoi, and W. Greene, Proc. Natl. Acad. Sci. USA 84:2002-2006, 1987). An affinity-purified antibody prepared against this peptide segment (p81) is shown here to cross-react with a homologous region of the human T-cell leukemia virus type I (HTLV-I) envelope glycoprotein, raising the interesting possibility that the envelope glycoprotein of HTLV-I can interact with the beta hIL-2 receptor.     

Dimethylsulfoxide-stabilized conformer of guanine-adenine repeat strand of DNA

Circular dichroism spectroscopy and other methods are used to show that the addition of dimethylsulfoxide causes reversible folding of the (GA)(10) strand of DNA into an ordered single-stranded conformer. The ordered conformer melts in a cooperative way and it does not contain protonated adenine. The (TA)(10), (A)(20), and (G)(20) are all unstable in this conformer. To the best of our knowledge, this is the first known ordered conformer of DNA that is stabilized by dimethylsulfoxide. This conformer might be a DNA analog of the protein alpha helix, which is an interesting idea for thinking about the evolution of DNA.     

General strategy for decoration of enveloped viruses with functionally active lipid-modified cytokines

Viral particles preferentially incorporate extra- and intracellular constituents of host cell lipid rafts, a phenomenon central to pseudotyping. Based on this mechanism, we have developed a system for the predictable decoration of enveloped viruses with functionally active cytokines that circumvents the need to modify viral proteins themselves. Human interleukin-2 (hIL-2), hIL-4, human granulocyte-macrophage colony-stimulating factor (hGM-CSF), and murine IL-2 (mIL-2) were used as model cytokines and fused at their C terminus to the glycosylphosphatidylinositol (GPI) acceptor sequence of human Fcgamma receptor III (CD16b). We show here that genetically modified cytokines are all well expressed on 293 producer cells. However, only molecules equipped with GPI anchors but not those linked to transmembrane/intracellular regions of type I membrane proteins are efficiently targeted to lipid rafts and consequently to virus-like particles (VLP) induced by Moloney murine leukemia virus Gag-Pol. hIL-4::GPI and hGM-CSF::GPI coexpressed on VLP were found to differentiate monocytes towards dendritic cells. Apart from myeloid-committed cell types, VLP-bound cytokines also act efficiently on lymphocytes. hIL-2::GPI strongly costimulated T-cell receptor (TCR)/CD3 dependent T-cell activation in vitro and mIL-2::GPI-coactivated antigen-specific T cells in vivo. On a molar basis, the functional activity of VLP-bound hIL-2::GPI was found to be comparable to that of soluble hIL-2. VLP decorated with hIL-2::GPI and coexpressing a TCR/CD3 ligand have an IL-2-specific activity of 5 x 10(4) units/mg protein. Virus particles decorated with lipid-modified cytokines might help to improve viral strains for vaccination purposes, the propagation of factor-dependent cell types, as well as gene transfer by viral systems in the future.     

Structure-function analysis of human IL-6: identification of two distinct regions that are important for receptor binding.

Interleukin-6 (IL-6) is a multifunctional cytokine that plays an important role in host defense. It has been predicted that IL-6 may fold as a 4 alpha-helix bundle structure with up-up-down-down topology. Despite a high degree of sequence similarity (42%) the human and mouse IL-6 polypeptides display distinct species-specific activities. Although human IL-6 (hIL-6) is active in both human and mouse cell assays, mouse IL-6 (mIL-6) is not active on human cells. Previously, we demonstrated that the 5 C-terminal residues of mIL-6 are important for activity, conformation, and stability (Ward LD et al., 1993, Protein Sci 2:1472-1481). To further probe the structure-function relationship of this cytokine, we have constructed several human/mouse IL-6 hybrid molecules. Restriction endonuclease sites were introduced and used to ligate the human and mouse sequences at junction points situated at Leu-62 (Lys-65 in mIL-6) in the putative connecting loop AB between helices A and B, at Arg-113 (Val-117 in mIL-6) at the N-terminal end of helix C, at Lys-150 (Asp-152 in mIL-6) in the connecting loop CD between helices C and D, and at Leu-178 (Thr-180 in mIL-6) in helix D. Hybrid molecules consisting of various combinations of these fragments were constructed, expressed, and purified to homogeneity. The conformational integrity of the IL-6 hybrids was assessed by far-UV CD. Analysis of their biological activity in a human bioassay (using the HepG2 cell line), a mouse bioassay (using the 7TD1 cell line), and receptor binding properties indicates that at least 2 regions of hIL-6, residues 178-184 in helix D and residues 63-113 in the region incorporating part of the putative connecting loop AB through to the beginning of helix C, are critical for efficient binding to the human IL-6 receptor. For human IL-6, it would appear that interactions between residues Ala-180, Leu-181, and Met-184 and residues in the N-terminal region may be critical for maintaining the structure of the molecule; replacement of these residues with the corresponding 3 residues in mouse IL-6 correlated with a significant loss of alpha-helical content and a 200-fold reduction in activity in the mouse bioassay. A homology model of mIL-6 based on the X-ray structure of human granulocyte colony-stimulating factor is presented.     

Cysteine-scanning mutagenesis and substituted cysteine accessibility analysis of transmembrane segment 4 of the Glut1 glucose transporter

A low resolution model has been proposed for the exofacial conformation of the Glut1 glucose transporter in which eight transmembrane segments form an inner helical bundle stabilized by four outer helices. The role of transmembrane segment 4, predicted to be an inner helix in this structural model, was investigated by cysteine-scanning mutagenesis in conjunction with the substituted cysteine accessibility method using the membrane-impermeant, sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS). A functional, cysteine-less, parental Glut1 molecule was used to produce 21 Glut1 point mutants by individually changing each residue along transmembrane helix 4 to a cysteine. The single cysteine mutants were then expressed in Xenopus oocytes, and their expression levels, transport activities, and sensitivities to pCMBS were determined. In striking contrast to all of the other seven predicted inner helices, none of the 21 helix 4 single-cysteine mutants was demonstrably inhibited by pCMBS. However, cysteine substitution within helix 4 resulted in an unusually high number of severely transport-defective mutants. The low absolute transport activities of two of these mutants (G130C and G134C) were due to their extremely low levels of expression, presumably a result of structural instability and consequent degradation in oocytes, suggesting that these two residues play an important role in maintaining the native structure of Glut1. The other two transport-defective mutants (Y143C and E146C) exhibited low specific transport activities, implying that these two residues play an important role in the transport cycle. Based on these data, we conclude that the exoplasmic end of helix 4 lies outside the inner helical bundle in the exofacial configuration of Glut1.     

Exendin-4 and glucagon-like-peptide-1: NMR structural comparisons in the solution and micelle-associated states.

Exendin-4, a 39 amino acid peptide originally isolated from the oral secretions of the lizard Heloderma suspectum, has been shown to share certain activities with glucagon-like-peptide-1 (GLP-1), a 30 amino acid peptide. We have determined the structuring preferences of exendin-4 and GLP-1 by NMR in both the solution and dodecylphosphocholine (DPC) micelle-associated states. Based on both chemical shift deviations and the pattern of intermediate range NOEs, both peptides display significant helicity from residue 7 to residue 28 with greater fraying at the N-terminus. Thornton and Gorenstein [(1994) Biochemistry 33, 3532-3539] reported that the presence of a flexible, helix-destabilizing, glycine at residue 16 in GLP-1 was an important feature for membrane and receptor binding. Exendin-4 has a helix-favoring glutamate as residue 16. In the micelle-associated state, NMR data indicate that GLP-1 is less helical than exendin-4 due to the presence of Gly16; chemical shift deviations along the peptide sequence suggest that Gly16 serves as an N-cap for a second, more persistent, helix. In 30 vol-% trifluoroethanol (TFE), a single continuous helix is evident in a significant fraction of the GLP-1 conformers present. Exendin-4 has a more regular and less fluxional helix in both media and displays stable tertiary structure in the solution state. In the micelle-bound state of exendin-4, a single helix (residues 11-27) is observed with residues 31-39 completely disordered and undergoing rapid segmental motion. In aqueous fluoroalcohol or aqueous glycol, the Leu21-Pro38 span of exendin-4 forms a compact tertiary fold (the Trp-cage) which shields the side chain of Trp25 from solvent exposure and produces ring current shifts as large as 3 ppm. This tertiary structure is partially populated in water and fully populated in aqueous TFE. The Leu21-Pro38 segment of exendin-4 may be the smallest protein-like folding unit observed to date. When the Trp-cage forms, fraying of the exendin-4 helix occurs exclusively from the N-terminus; backbone NHs for the C-terminal residues of the helix display H/D exchange protection factors as large as 10(5) at 9 degrees C. In contrast, no tertiary structure is evident when exendin-4 binds to DPC micelles. An energetically favorable insertion of the tryptophan ring into the DPC micelle is suggested as the basis for this change. With the exception of exendin-4 in media containing fluoro alcohol cosolvents, NMR structure ensembles generated from the NOE data do not fully reflect the conformational averaging present in these systems. Secondary structure definition from chemical shift deviations may be the most appropriate treatment for peptides that lack tertiary structure.     

Membrane structure of bombesin studied by infrared spectroscopy. Prediction of membrane interactions of gastrin-releasing peptide, neuromedin B, and neuromedin C

Bombesin, in contact with flat phospholipid bilayer membranes, was shown to adopt a membrane structure similar to that of substance P, dynorphin-(1-13)-tridecapeptide, and adrenocorticotropin-(1-24)-tetracosapeptide. The C-terminal message segment, comprising 8-10 amino acid residues, is inserted into a relatively hydrophobic membrane compartment as an alpha-helical domain oriented perpendicularly on the membrane surface. The N-terminal, hydrophilic tetrapeptide segment remains in the aqueous compartment as a random coil. This was shown with IR and IR attenuated total reflection spectroscopy. Equilibrium thermodynamic estimations confirmed the observed membrane structure with respect to helix length, strength of hydrophobic membrane association, and orientation (caused by favorably oriented molecular amphiphilic and helix electric dipole moments). The membrane structure may explain why Trp-8 and His-12 are essential for biologic activity. Neuromedin B is predicted to be able to adopt a membrane structure similar to that of bombesin. However, gastrin-releasing peptide and neuromedin C are predicted not to behave in the same manner. The molecular mechanism of receptor subtype selection by bombesin-like peptides may prove to be similar to that observed earlier for opioid peptides and the neurokinins.     

Molecular mechanisms of cytokine receptor activation

Cytokine receptors are transmembrane proteins that transmit a signal into the cell upon ligand binding. Commonly, these molecules have one hydrophobic segment of about 20-26 amino acids that is believed to span the membrane as a helix and this divides these receptors into extra- and intracellular components. By utilizing two different epitopes, the cytokines bridge two receptor chains, resulting in a close proximity of the intracellular component and thereby initiating the intracellular signalling cascade. The dimerization event is believed to be the mechanism by which the signal is transmitted across a membrane. In the light of new results obtained for the erythropoietin receptor, James A. Wells questioned whether any dimer would be sufficient. This review will expand upon the above question by discussing the more complex signal-transducing receptor subunits of the Interleukin-6 type family of cytokines. Based on the recently solved quaternary structure of the Insulin receptor, possible analogies will be confronted.     

Structural analysis of peptide helices containing centrally positioned lactic acid residues

The effect of insertion of lactic acid (Lac) residues into peptide helices has been probed using specifically designed sequences. The crystal structures of 11-residue and 14-residue depsipeptides Boc-Val-Val-Ala-Leu-Val-Lac-Leu-Aib-Val-Ala-Leu-OMe (1) and Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-Val-Lac-Leu-Aib-Val-Ala-Leu-OMe (3), containing centrally positioned Lac residues, have been determined. The structure of an 11-residue peptide Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-Aib-Val-Ala-Leu-OMe (2), analog of a which is an amide previously determined Lac-containing depsipeptide, Boc-Val-Ala-Leu-Aib-Val-Lac-Leu-Aib-Val-Ala-Leu-OMe I. L. Karle, C. Das, and P. Balaram, Biopolymers, Vol. 59, (2001) pp. 276-289], is also reported. Peptide 1 adopts a helical fold, which is stabilized by mixture of 4-->1 and 5-->1 hydrogen bonds. Peptide 2 adopts a completely alpha-helical conformation stabilized by eight successive 5-->1 hydrogen bonds. Peptide 3 appears to be predominately alpha-helical, with seven 5-->1 hydrogen bonds and three 4-->1 interaction interspersed in the sequence. In the structure of peptide 3 in addition to water molecules in the head-to-tail region, hydration at an internal segment of the helix is also observed. A comparison of five related peptide helices, containing a single Lac residue, reveals that the hydroxy acid can be comfortably accommodated at interior positions in the helix, with the closest C=O...O distances lying between 2.8 and 3.3 A.     

Receptors for interleukin-13 and interleukin-4 are complex and share a novel component that functions in signal transduction.

Interleukin-4 (IL-4) and interleukin-13 (IL-13) are two cytokines that are secreted by activated T cells and have similar effects on monocytes and B cells. We describe a mutant form of human interleukin-4 (hIL-4) that competitively antagonizes both hIL-4 and human interleukin-13 (hIL-13). The amino acid sequences of IL-4 and IL-13 are approximately 30% homologous and circular dichroism (CD) spectroscopy shows that both proteins have a highly alpha-helical structure. IL-13 competitively inhibited binding of hIL-4 to functional human IL-4 receptors (called hIL-4R) expressed on a cell line which responds to both hIL-4 and IL-13. Binding of hIL-4 to an hIL-4 responsive cell line that does not respond to IL-13, and binding of hIL-4 to cloned IL-4R ligand binding protein expressed on heterologous cells, were not inhibited by IL-13. hIL-4 bound with approximately 100-fold lower affinity to the IL-4R ligand binding protein than to functional IL-4R. The mutant hIL-4 antagonist protein bound to both IL-4R types with the lower affinity. The above results demonstrate that IL-4 and IL-13 share a receptor component that is important for signal transduction. In addition, our data establish that IL-4R is a complex of at least two components one of which is a novel affinity converting subunit that is critical for cellular signal transduction.     

Mapping of the leptin binding sites and design of a leptin antagonist

The leptin/leptin receptor system shows strong similarities to the long-chain cytokine interleukin-6 (IL-6) and granulocyte colony-stimulating factor cytokine/receptor systems. The IL-6 family cytokines interact with their receptors through three different binding sites I-III. The leptin structure was superposed on the crystal structures of several long-chain cytokines, and a series of leptin mutants was generated focusing on binding sites I-III. The effect of the mutations on leptin receptor (LR) signaling and on binding to the membrane proximal cytokine receptor homology domain (CRH2) of the LR was determined. Mutations in binding site I at the C terminus of helix D show a modest effect on signaling and do not affect binding to CRH2. Binding site II is composed of residues at the surface of helices A and C. Mutations in this site impair binding to CRH2 but have only limited effect on signaling. Site III mutations around the N terminus of helix D impair receptor activation without affecting binding to CRH2. We identified an S120A/T121A mutant in binding site III, which lacks any signaling capacity, but which still binds to CRH2 with wild type affinity. This leptin mutant behaves as a potent leptin antagonist both in vitro and in vivo.     

A bioactive somatostatin analog without a type II' beta-turn: synthesis and conformational analysis in solution.

A cyclic somatostatin analog [structure: see text] (1) has been synthesized. Biological assays show that this compound has strong binding affinities to somatostatin hsst2 and hsst5 receptor subtypes (5.2 and 1.2 nM, respectively, and modest affinity to hsst4 (41.1 nM)). Our conformational analysis carried out in DMSO-d6 indicates that this compound exists as two structures arising from the trans and cis configurations of the peptide bond between Phe7 and N-alkylated Gly8. However, neither conformer exhibits a type II' beta-turn. This is the first report of a potent bioactive somatostatin analog that does not exhibit a type II' beta-turn in solution. Molecular dynamics simulations (500 ps) carried out at 300 K indicate that the backbone of compound 1 is more flexible than other cyclic somatostatin analogs formed by disulfide bonds.     

Internal deletions in human interleukin-6: structure-function analysis

By cDNA mutagenesis, we have constructed internal and C-terminal deletions (delta 21-51, delta 52-97, delta 97-104, delta 127-174, delta 97-184 and delta 134-184) in human interleukin-6 (hIL-6). All those deletion-carrying hIL-6 (delta hIL-6) proteins were then produced in Xenopus laevis oocytes and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results show that, at least in frog oocytes, the first potential N-glycosylation site (Asn45) is utilized exclusively. The IL-6 conformation of these deletion-carrying proteins has been studied by immunoprecipitation with two kinds of monoclonal antibodies (mAb's): mAb's that show preference towards denatured hIL-6, or conformation-specific mAb's. The binding pattern of these two series of mAb's indicated that the IL-6 conformation has been largely destroyed for four of our delta-proteins. Proteins delta 21-51 and delta 127-174 have kept a part of the IL-6 tertiary structure since they are still recognized by some conformation-specific mAb's. All of these delta hIL-6 proteins were inactive in the IL-6 hybridoma growth factor (HGF) assay and unable to inhibit the HGF activity of the recombinant human wild-type IL-6 (wt hIL-6). Moreover, the oocyte-synthesized delta hIL-6 (delta 21-51, delta 127-174, delta 97-184, delta 134-184) did not bind to the IL-6 receptor. Finally, we have produced two proteins with aa 29-33 or 97-104 substituted by corresponding murine IL-6 (mIL-6) sequences.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure-activity relationships of interleukin-8 determined using chemically synthesized analogs. Critical role of NH2-terminal residues and evidence for uncoupling of neutrophil chemotaxis, exocytosis, and receptor binding activities.

Interleukin-8 (IL-8) is an inflammatory mediator that stimulates neutrophil migration and functional activation. Analogs of human IL-8 were chemically synthesized, purified, and compared with the full-length 72-residue synthetic IL-8 for their ability to stimulate neutrophil chemotaxis and exocytosis as measured by assaying for release of elastase, as well as their binding to specific receptors in competition assays. Analogs corresponding to the less abundant natural forms, 3-72, 4-72, and 77-residue IL-8, were evaluated and the 3-72 and 4-72 had 2-5-fold higher potencies, whereas the 77-residue IL-8 was 2-fold less potent. A major finding was that NH2-terminal residues 4, 5, and 6 were absolutely essential for IL-8 activity and receptor binding. Quantitative dissociation of elastase release and chemotaxis activity was detected with 5-72, which compared with 1-72, was 80-fold less potent in the elastase assay, but was only slightly less potent in stimulating chemotaxis. IL-8 6-72 lacked all the biological activities tested but had detectable receptor binding activity. The NH2-terminal peptide, AVLPRSAKEL, lacked activity and receptor binding, suggesting that the NH2-terminal region alone is not sufficient for function. Comparison of analogs shortened at the COOH terminus showed that potency was progressively reduced as the COOH-terminal residues were excluded. However activity was retained in an analog (1-51) with the entire COOH-terminal alpha helix and beta turn missing. A peptide corresponding to the COOH-terminal 22 residues, although inactive alone, synergized with the 1-51 analog in stimulating elastase release. The results suggest that the NH2-terminal residues 4, 5, and 6, which are disordered in the IL-8 solution structure, are directly involved in receptor binding, but the COOH-terminal alpha helix is probably important for stabilizing the three-dimensional structure. Other regions within residues 7-51 are also functionally important.     

Structure of a receptor-binding fragment from human luteinizing hormone beta-subunit determined by [1H]- and [15N]nuclear magnetic resonance spectroscopy.

The structure of the glycoprotein hormones (LH, CG, FSH, and TSH) and their mechanism of receptor recognition are problems of long-standing interest and speculation. Here we describe the two-dimensional [1H]nuclear magnetic resonance ([1H]NMR) analysis of a linear peptide model for the intercysteine sequence (38-57) from the beta-subunit of human (h) LH. This sequence contains functional determinants for receptor binding and postreceptor activation and is predicted by computer-based modeling to fold as a compact minidomain containing a central amphipathic helix. To test this prediction, an Arg-extended disulfide-free (38-57) analog of enhanced solubility was prepared for complementary circular-dichroic and two-dimensional NMR studies. The linear peptide retains ovarian membrane receptor-binding activity. Although the peptide is not highly structured in aqueous solution, circular-dichroic analysis shows partial alpha-helix formation in a lipophilic medium (50% trifluoroethanol). Complete sequential assignment is obtained in 50% trifluoroethanol based on homonuclear and [15N]edited heteronuclear NMR methods. alpha-Helix-related (i,i + 3) connectivities are observed by nuclear-Overhauser effect spectroscopy that define an amphipathic alpha-helical segment (residues 41-48). Additional long range nuclear-Overhauser effects are observed in the C-terminal region that are consistent with beta-turns involving one or more proline residues; these may serve to reverse the direction of the peptide chain. A nuclear-Overhauser effect contact is identified between residues 38 and 55 at opposite ends of the linear sequence, suggesting that a loop configuration is significantly populated in this solvent system. These results, taken together, characterize elements of ordered structure in the 38-57 peptide, which appear to be distinguishing features of hLH (and the homologous region of hCG). We propose that the structure of this peptide provides a model for the structure of the corresponding region of native hLH in the hormone-receptor complex.     

Generation of highly stable IL-18 based on a ligand-receptor complex structure

Human interleukin-18 (hIL-18), initially cloned as an IFN-gamma-inducing factor, has a key role in many inflammatory diseases. We have previously developed a high production system for correctly folded active hIL-18 protein, leading to the revelation of the 3D-structure and the receptor binding mode. These findings can strongly indicate the experimental and medical applications of IL-18; however, the recombinant protein is prone to be inactivated forming multimers. Recently, therapeutic approaches using recombinant IL-18 have shown the effectiveness for treatment of cancer; indicating the necessity of a more stable protein for therapy with intertrial reliability. Here we have generated a highly stable hIL-18 with replacement of cysteine by serine based on the tertiary structure and the binding mechanism, retaining the biological activity. Similar rational designs can be applied to develop new therapeutic molecules of other cytokines.     

High Yield Production of a Soluble Human Interleukin-3 Variant from E. coli with Wild-Type Bioactivity and Improved Radiolabeling Properties

Human interleukin-3 (hIL-3) is a polypeptide growth factor that regulates the proliferation, differentiation, survival and function of hematopoietic progenitors and many mature blood cell lineages. Although recombinant hIL-3 is a widely used laboratory reagent in hematology, standard methods for its preparation, including those employed by commercial suppliers, remain arduous owing to a reliance on refolding insoluble protein expressed in E. coli. In addition, wild-type hIL-3 is a poor substrate for radio-iodination, which has been a long-standing hindrance to its use in receptor binding assays. To overcome these problems, we developed a method for expression of hIL-3 in E. coli as a soluble protein, with typical yields of >3mg of purified hIL-3 per litre of shaking microbial culture. Additionally, we introduced a non-native tyrosine residue into our hIL-3 analog, which allowed radio-iodination to high specific activities for receptor binding studies whilst not compromising bioactivity. The method presented herein provides a cost-effective and convenient route to milligram quantities of a hIL-3 analog with wild-type bioactivity that, unlike wild-type hIL‑3, can be efficiently radio-iodinated for receptor binding studies.