©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structure-Function Relationships in Human Interleukin-11
IDENTIFICATION OF REGIONS INVOLVED IN ACTIVITY BY CHEMICAL MODIFICATION AND SITE-DIRECTED MUTAGENESIS (*)

(Received for publication, August 15, 1994; and in revised form, November 4, 1994)

Marta J. Czupryn (§) John M. McCoy Hubert A. Scoble

From the Genetics Institute, Andover, Massachusetts 01810

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Chemical modification approaches combined with site-directed and deletion mutagenesis have been used to identify functionally critical regions/residues of recombinant human IL-11 (rhIL-11). Incubation of rhIL-11 with iodoacetic acid results in specific alkylation of a single methionine residue, Met, and a 25-fold reduction of in vitro biological activity on mouse plasmacytoma cells. A similar decrease in activity is observed when Met is substituted with Ala, Leu, Gln, Glu, or Lys by site-directed mutagenesis. Treatment of rhIL-11 with succinic anhydride leads to modification of the amino-terminal amino group and partial labeling of 2 lysines, Lys and Lys, and to a 3-fold decrease in activity. The activity losses can be attributed to modification of the lysine residues, since the succinyl derivative of the amino terminus is fully active. In addition, carboxyl-terminal deletion mutagenesis studies have demonstrated that removal of the last 4 residues reduces rhIL-11 activity 25-fold, whereas removal of 8 or more amino acids results in an inactive molecule. Based on secondary structure predictions and the location of exon/intron boundaries in the IL-11 genomic structure, we propose a four-helix bundle topology as a structural model for rhIL-11. This model has been tested by limited proteolysis using three side chain-specific endoproteinases. A limited number of protease-sensitive cleavage sites are present in rhIL-11, and all but two are located in the postulated helix interconnecting loops or at helix termini. alpha-Helices, which in the proposed structure form a compact core of the molecule, are inaccessible to digestion under limiting conditions. According to the model, Met, Lys and Lys are located on the surface of the molecule, in agreement with their preferential accessibility to chemical modification. By analogy with human growth hormone, we postulate that Met and the carboxyl terminus of rhIL-11 are involved in the primary receptor binding site (site I), whereas Lys and Lys may be a part of binding site II.


INTRODUCTION

Interleukin-11 (IL-11) (^1)is a multifunctional cytokine, which affects growth and differentiation of several hematopoietic cell types, including early pluripotent stem cells, megakaryocyte progenitors and megakaryocytes, erythrocyte progenitors, and granulocyte progenitors (for a review, see Refs. 1 and 2). The biological effects of IL-11, however, are not limited to cells of hematopoietic origin. IL-11 has been reported to inhibit adipogenesis in preadipocytes (3) and to stimulate production of several acute phase plasma proteins in hepatocytes (4) and the tissue inhibitor of metalloproteinases in connective tissue cells(5) . Some of the biological activities of IL-11 overlap with those of interleukin-6 (IL-6), even though these two cytokines are biochemically distinct(6) . It has recently been demonstrated that the signal-transducing subunit of the IL-6 receptor complex, gp130, is involved in the signaling pathway of IL-11(7) . Additionally, these two cytokines can activate expression of the same early response genes(7) , which may, in part, explain their common biological functions. However, the ligand binding subunits of the IL-6 and IL-11 receptors are different. IL-6 interacts with an 80-kDa ligand-binding protein p80, expressed on target cells (8) , which then associates with the signal-transducing, non-ligand-binding subunit gp130 and triggers activation of the intracellular signal transduction pathway(9) . Initial characterization of the IL-11 receptor by affinity cross-linking studies on mouse 3T3-L1 preadipocytes suggests that the IL-11 binding receptor subunit is different from p80 and consists of a single polypeptide chain of 151 kDa(10) .

There is no information available about the tertiary fold of IL-11. Recent structural studies by x-ray crystallography or multidimensional NMR of several members of the cytokine family have revealed a common structural motif, consisting of a bundle of four alpha-helices connected by loops of variable length (reviewed in (11) and (12) ). To date, the four-helix bundle fold has been observed in growth hormone (GH)(13, 14) , interleukin-2 (IL-2)(15, 16) , interleukin-4 (IL-4)(17, 18, 19, 20) , granulocyte colony-stimulating factor(21) , granulocyte-macrophage colony-stimulating factor(22, 23) , as well as in macrophage colony-stimulating factor (M-CSF) (24) and interleukin-5 (IL-5)(25) , which are both dimeric molecules. It has been proposed that IL-11 and IL-6 share this same basic architecture(11) . In the case of IL-6, two models of the tertiary fold predicted by comparative molecular modeling have been suggested(26, 27) , but its structure has not yet been solved.

The availability of recombinant human IL-11 has enabled us to initiate structure-function studies aimed at identifying structural features which are essential for the biological function of this molecule. Using selective chemical modifications, combined with deletion and site-directed mutagenesis, we show that two regions of rhIL-11 are important for its activity. These regions have analogous locations within the molecule as the receptor binding sites on hGH (14) and could potentially play a role in the interaction of IL-11 with its receptor complex. To further understand the structure and functional properties of IL-11 and the interaction with its receptor, we have constructed a model of the tertiary structure of IL-11. We have tested this model using limited proteolysis as a probe for distinct structural motifs.


MATERIALS AND METHODS

rhIL-11

Recombinant hIL-11 (des-Pro hIL-11) contains 177 amino acid residues and has a molecular mass of 19,047 Da. It is expressed in Escherichia coli as part of a fusion protein with thioredoxin, from which it is cleaved and purified to homogeneity using conventional chromatography(28) .

Mutagenesis

rhIL-11 mutants were constructed using a thioredoxin-hIL-11 fusion expression vector based on pTRXFUS(28) . Specific point mutations or deletions in the rhIL-11 coding sequence were constructed by replacing short segments of the gene, between flanking restriction sites, with synthetic DNA duplexes containing the desired changes. For each mutant, the sequence of the introduced synthetic oligonucleotide section was confirmed by DNA sequence analysis. Fusion proteins were expressed in E. coli K12 strain GI724 (ATCC 55151) and released from cells and purified as described previously(28) .

Chemical Modifications

rhIL-11 (50 µM) was carboxymethylated by treatment with 60 mM iodoacetic acid in 50 mM sodium acetate buffer, pH 5.5, for 4 h at 20 °C. Chemically modified rhIL-11 was separated from the excess reagent and the residual unmodified protein by reversed-phase HPLC, using a Vydac C4 column (0.46 times 25 cm) and a linear gradient of 20-70% (v/v) acetonitrile in 0.1% trifluoroacetic acid (w/v) over 100 min, at a flow rate of 0.75 ml/min. Fractions containing alkylated material were collected and concentrated by vacuum centrifugation.

Succinylated derivatives were prepared by incubating rhIL-11 (50 µM) in 0.2 M MOPS, pH 8.0, with 0.1-1 mM succinic anhydride. Succinic anhydride was prepared as a stock solution (0.5 M) in dimethylformamide and diluted in 0.2 M MOPS, pH 8.0, immediately before use. The reaction was carried out for 30 min at room temperature, and the pH was maintained at 8.0 by adding 0.1 M NaOH. Succinylated rhIL-11 forms were purified by reversed-phase chromatography as described above.

Carboxyl-terminally Truncated rhIL-11

Carboxyl-terminally truncated rhIL-11 (residues 1-133) was generated by incubating the protein in 0.1% trifluoroacetic acid at 50 °C for 5 h. Truncated rhIL-11 was separated from the intact molecule and the carboxyl-terminal fragment 134-177 by reversed-phase HPLC on a Vydac C4 column using the conditions described for alkylated rhIL-11. To confirm the identity of the fragments, samples were analyzed by amino-terminal sequencing and MALDI-TOF mass spectrometry.

Structural Characterization of rhIL-11

Size exclusion HPLC analyses were performed using a TSK G2000SW column (0.78 times 30 cm, The Nest Group, Inc., Southboro, MA). Proteins were eluted with 0.5 M NaCl, 20 mM MES, pH 6.0, at a flow rate of 0.7 ml/min. Absorbance was monitored at 225 and 280 nm.

For cation exchange HPLC, a Bakerbond wide-bore CBX column was used (0.46 times 5 cm, J.T. Baker) at a flow rate of 2 ml/min. Buffer A contained 20 mM glycine, pH 9.0, buffer B was 0.5 M NaCl, 20 mM glycine, pH 9.0. For protein elution, a linear gradient of 5 to 50% buffer B in 10 min was employed. Absorbance was monitored at 225 and 280 nm.

SDS-PAGE was performed as described by Laemmli (29) in 17-27% polyacrylamide gradient pre-cast gels (ISS, Natick, MA). The gels were stained using ammoniacal silver stain kit (ISS). rhIL-11 was observed to migrate during SDS-PAGE with an apparent molecular mass of 22 kDa. For native PAGE, a 4% stacking gel cast on top of a 12.5% resolving gel was used and a continuous buffer system containing 30 mM histidine, 40 mM MES, pH 6.0. The gels were run at 8 V/cm for 3 h and stained with Coomassie blue.

Proteolytic Digestion

All enzymes used were of sequencing grade (Boehringer Mannheim). Trypsin and chymotrypsin digestion of rhIL-11 was carried out in 50 mM ammonium bicarbonate at the enzyme:substrate ratio of 1:20 (w/w) and 1:50 (w/w), respectively. Digests were incubated at 37 °C under argon for the indicated periods of time, and proteolysis was stopped by adding 3 µl of trifluoroacetic acid and 5 µl of heptafluorobutyric acid to the 1-ml digest solution.

Endoproteinase Asp-N digestion was carried out in 50 mM Tris-HCl, pH 7.5, at room temperature, using an enzyme:substrate ratio of 1:50 (w/w). Limited proteolysis was carried out at room temperature for 10-30 min. Complete digestion was achieved by incubating the protein with enzyme at 37 °C for 19-20 h.

Reversed-phase Chromatography of rhIL-11 Peptides

The rhIL-11 digests were injected onto a Vydac C18 column (0.46 times 25 cm), and peptides were eluted with a multistep linear gradient using 4 mM heptafluorobutyric acid, 6 mM trifluoroacetic acid as solvent A and 4 mM heptafluorobutyric acid, 6 mM trifluoroacetic acid in 95% acetonitrile as solvent B, at the flow rate of 0.75 ml/min. Gradient steps were as follows: 0-10 min, 5% B; 10-55 min, 5-35% B; 55-85 min, 35-50% B; 85-100 min, 50-65% B; 100-105 min, 65-90% B; and 105-115 min, 90% B. Absorbance was monitored at 214 and 280 nm. Peak fractions containing rhIL-11 peptides were collected manually and stored at -20 °C for subsequent analysis.

Amino-terminal Sequencing

rhIL-11 peptides were subjected to automated Edman degradation using an ABI model 475A gas-phase sequencing system equipped with an on-line ABI 120A phenylthiohydantoin amino acid detector. Prior to analysis, peptide-containing HPLC fractions were concentrated by vacuum centrifugation to a volume less than 50 µl.

Amino Acid Analysis

Alkylated and control rhIL-11 samples were hydrolyzed in vacuo with 6 N constant boiling HCl at 110 °C for 24 h. The hydrolysate was analyzed by the PicoTag^R method (30) using pre-column derivatization with phenylisothiocyanate.

Fast Atom Bombardment Mass Spectrometry

FAB MS analysis of the HPLC fractions containing rhIL-11 peptides was carried out using the first two sectors of a JEOL HX110/HX110 high resolution tandem mass spectrometer. The instrument was scanned from m/z 0 to 8000 at a resolution of 1:3000. The accelerating voltage was 10 kV, and the instrument was calibrated using CsI cluster ions. Prior to analysis, fractions were combined with 100 µl of 2% aqueous glycerol and concentrated in vacuo. A 0.5-1.0-µl aliquot of this solution was mixed with 0.1-0.3 µl of monothioglycerol and placed onto the FAB probe for analysis.

Matrix-assisted Laser Desorption Ionization Time-of-flight Mass Spectrometry

MALDI-TOF analysis of the peptide fractions was performed using the Bruker Reflex time-of-flight mass spectrometer. The samples were analyzed in the linear mode with 30 kV accelerating voltage, and the instrument was calibrated using adrenocorticotropic hormone 18-39 (m/z 2466.7) and E. coli thioredoxin (m/z 11674.4) as standards. A 1-µl aliquot of the sample was mixed with 2 µl of a saturated solution of alpha-cyano-4-hydroxycinnamic acid matrix (5 mg/ml) in 0.1% trifluoroacetic acid (w/v), 30% acetonitrile (v/v). The sample-matrix solution was placed onto the MALDI-TOF probe and air-dried prior to analysis.

CD Spectroscopy

Far-UV CD spectra were recorded on a JASCO J-600 spectropolarimeter at 25 °C using a quartz cell of 0.01 cm pathlength. The instrument was calibrated with (+)-10-camphorsulfonic acid as described by Johnson(31) . The wild type rhIL-11 and M58A mutant were equilibrated in 20 mM sodium phosphate buffer, pH 7.0, and the protein concentration was adjusted to 0.5 and 0.3 mg/ml, respectively. Spectra are expressed in units of Delta (litersbulletcmbullet(mol of residue)), where Delta is the difference between the extinction coefficients for left and right circularly polarized light and were calculated based on a mean residue weight of 107.5 for each protein.

Biological Assays

In vitro biological activity of the native, modified, and truncated rhIL-11 forms and M58A mutant protein was assessed in a proliferation assay on a mouse plasmacytoma cell line T10(32) . Protein concentrations were determined by amino acid analysis as described above. When crude E. coli lysates were used in the assay, relative concentrations of mutant-thioredoxin fusion protein was estimated by scanning Coomassie Blue-stained gels.

Structure Predictions

Predictions of secondary structure of rhIL-11 were performed according to Chou and Fasman (33) and Garnier et al.(34) using the GCG computer program package. The length of the alpha-helices was determined from helical wheel projections based on the preservation of amphipathic character.


RESULTS AND DISCUSSION

Initial screening of functionally important amino acid residues in rhIL-11 involved the combined use of side chain-selective chemical modifications and site-directed mutagenesis. Iodoacetic acid and succinic anhydride were selected as modifying reagents for these studies, since rhIL-11 contains only six potential alkylation sites (two methionines and four histidines) and four free amino groups that could be succinylated (amino terminus and three lysines) (Fig. 1).


Figure 1: Amino acid sequence of rhIL-11. Potential alkylation sites are denoted by circles, potential succinylation sites by squares. Peptides expected from endoproteinase Asp-N cleavage are underlined and numbered D1 through D12.



Modification of Methionine Residues

Modification of rhIL-11 with iodoacetic acid was carried out at pH 5.5 to selectively target methionine, but not histidine, residues. One major derivative was obtained, which eluted from the C4 column with a retention time that was earlier than that of the native rhIL-11 (Fig. 2A). Two minor components could also be detected (indicated by arrows in Fig. 2A), but the amount did not exceed 5% of the total material, even upon prolonged incubation with iodoacetic acid. Amino acid analysis of the alkylated rhIL-11 demonstrated loss of 1 out of the 2 methionine residues, but no modification of histidines (results not shown). To determine the site of modification, peptide maps of the native and alkylated rhIL-11 were generated using endoproteinase Asp-N. The enzyme would be expected to cleave rhIL-11 into 12 peptides (see Fig. 1), 2 of which contain methionine, peptides D8 (Met) and D10 (Met). As shown in Fig. 2B, all peaks in the modified sample appeared at the same elution positions as in nonalkylated rhIL-11, with the exception of one peptide, which was identified as D8 (see below). The retention time of this peptide was markedly decreased compared to the unmodified control sample, consistent with its more polar character after modification. The elution position of the other methionine-containing peptide, D10, was not affected by alkylation, indicating that position 58, not 122, was the site of modification. Modification of Met was supported by amino-terminal sequence analysis of the alkylated peptide D8, which showed a substantial loss of methionine signal at position 58 and the appearance in the same cycle of a new peak, eluting in a position which did not correspond to any of the known phenylthiohydantoin-amino acid derivatives (result not shown). FAB MS analysis of the modified D8 peptide resulted in a predominant signal at m/z 2751.5, which is consistent with the mass expected for the D8 peptide that has been modified by carboxymethylation (theoretical (M + H) 2751.4). Amino-terminal sequencing and FAB MS analysis of D10 peptide, derived from the sample treated with iodoacetic acid, did not detect any modification of Met, even after increasing the concentration of the alkylating agent or the incubation time. However, when the alkylation procedure was performed on the carboxyl-terminally truncated 1-133 rhIL-11 variant, Met was accessible to alkylation. Gel filtration chromatography of the truncated variant showed an increased hydrodynamic volume compared to the intact molecule, suggesting that removal of the carboxyl-terminal residues 134-177 resulted in protein unfolding, thereby exposing Met for modification (result not shown). These data suggest that Met is located on the surface of the molecule, whereas Met would be buried within the hydrophobic core of the protein.


Figure 2: Modification of rhIL-11 with iodoacetic acid. A, reversed-phase HPLC profiles of control rhIL-11 and rhIL-11 modified with 60 mM iodoacetic acid (IAA). 30 µg of protein was applied to the column. Elution positions of two minor modified products are indicated by arrows. B, endoproteinase Asp-N peptide maps of control and alkylated rhIL-11. Expected peptides are identified in the control sample and labeled according to Fig. 1.



The effect of Met alkylation on in vitro bioactivity of rhIL-11 was tested in the T10 proliferation assay. Modification of this single residue dramatically reduced the ability of rhIL-11 to stimulate proliferation of T10 cells (Table 1), suggesting that Met is involved in rhIL-11 function in vitro. To confirm this suggestion and to exclude the possibility that the reduced activity resulted from changes in tertiary structure of the molecule caused by chemical modification, methionine 58 substitution mutants were generated using site-directed mutagenesis. Initially, Met was replaced by alanine, since its small side chain would be expected to minimally perturb the protein structure. Moreover, in contrast to chemical modification, which converts the neutral methionine residue into a positively charged methionine carboxymethyl-sulfonium ion, methionine to alanine replacement does not affect the charge of the molecule. The M58A mutant was expressed in E. coli as a fusion protein with E. coli thioredoxin. The expression level and solubility of the mutant fusion protein was similar to the wild type rhIL-11-thioredoxin fusion, indicating that the amino acid replacement had no major effect on protein folding or stability. The M58A mutant was then purified to near-homogeneity, and the methionine to alanine replacement was confirmed by peptide mapping and amino-terminal amino acid analysis of the peptide D8 (results not shown). In the T10 bioassay, the M58A mutant was 7-fold less active than wild type rhIL-11 (Table 1). Decreased activity of M58A could not be attributed to effects of amino acid substitution on the global protein conformation, since the mutant protein showed behavior identical with that of the wild type rhIL-11 upon native and denaturing PAGE and size exclusion and cation exchange chromatography (results not shown). The far-UV CD spectra of the M58A mutant and rhIL-11 were also indistinguishable within experimental error (Fig. 3). Analysis of the CD spectrum for singular value decomposition and variable selection (31) suggests that wild type rhIL-11 and M58A mutant each contains 57-58% alpha-helical structure.




Figure 3: Far-UV CD spectra of the wild type rhIL-11 and M58A mutant.



To further investigate the role of Met in rhIL-11 function, multiple additional amino acid substitutions were generated at position 58. These mutants were not subjected to cleavage and purification, but were assayed for bioactivity as fusion proteins with thioredoxin using E. coli lysates. It has been demonstrated previously that linkage of rhIL-11 to thioredoxin does not affect the biological activity of the cytokine(28) . Based on the expression levels and solubility of the mutant fusion proteins, neither mutation affected gross protein structure. Substitution of Met by hydrophobic (leucine), noncharged polar (glutamine) or charged (glutamic acid and lysine) amino acids resulted in similar or even greater losses in in vitro activity than did substitution with alanine (Table 1). Introduction of the positively charged lysine residue at position 58 had the largest inhibitory effect, mimicking the effect of protein alkylation. These results suggest that the hydrophobic character of residue 58 is critical for biological activity of rhIL-11.

Modification of Lysine Residues

Reaction of rhIL-11 with the amino group-specific reagent succinic anhydride resulted in a progressive modification of the protein with increasing concentration of the reagent, as indicated by the appearance of new peaks with increased retention times upon HPLC analysis (Fig. 4A). Mass spectra of the succinylated derivatives were consistent with incorporation of one ((M + H) = 19,169.0) to six ((M + H) = 19,621.5) modifying groups per mol of rhIL-11, depending on the succinic acid concentration. Since there are only four amino groups in the molecule, this result indicates that other functional groups (e.g. hydroxyamino acids) were also modified by the excess amount of the reagent.


Figure 4: Modification of rhIL-11 with succinic anhydride. A, reversed-phase HPLC profiles of control rhIL-11 and rhIL-11 modified with 0.1 mM or 1.0 mM succinic anhydride (SA). 250 µg of protein was applied to the column. The number of succinyl groups incorporated per mol of protein was determined by MALDI-TOF MS analysis of the indicated HPLC fractions. B, endoproteinase Asp-N peptide maps of control and succinylated rhIL-11.



To determine which amino acid side chains were most accessible to modification at low reagent concentration, samples of succinylated rhIL-11 containing approximately one and two modifying groups per molecule were subjected to peptide mapping (Fig. 4B). The map of monosuccinylated rhIL-11 derivative revealed the appearance of a single new peak, which was identified as the modified amino-terminal peptide D1. The molecular mass of this peptide and lack of signal upon amino acid sequencing were consistent with succinylation of the amino-terminal amino group. Based on the peak area, modification was estimated to affect 85% of rhIL-11 molecules.

In the sample with two modifying groups incorporated per mol of protein, 3 residues were found to be succinylated (Fig. 4B). In addition to the amino-terminal amino group, which was fully succinylated in this sample, 35% of the total amount of peptide D5 and 50% of peptide D9 were covalently modified. Both of these peptides contain single lysine residues at positions 41 and 98, respectively, modification of which was confirmed by amino-terminal sequencing of the isolated peptides (results not shown).

In the T10 bioassay, amino-terminally modified rhIL-11 was fully active (Table 1). This result is in agreement with the fact that the fusion protein retains biological activity even though thioredoxin is linked to the amino terminus of rhIL-11 (28) and strongly suggests that the amino-terminal portion of the protein is not involved in rhIL-11 function. In contrast to the amino-terminal amino group modification, partial succinylation of the lysine residues at positions 41 and 98 resulted in loss of nearly two-thirds of rhIL-11 bioactivity (Table 1), indicating that Lys and/or Lys are important for in vitro function.

Role of the Carboxyl Terminus

The carboxyl-terminal portion of several cytokines, including hGH(14, 35) , IL-4(36, 37) , and IL-6 (38, 39) , has been shown to be critical for their activity and receptor binding. To assess the role of the carboxyl terminus in rhIL-11 function, a series of deletion mutants were generated. Segments of 4 residues, each corresponding to approximately one helical turn, were deleted sequentially, resulting in five mutant proteins lacking 4, 8, 12, 16, and 20 carboxyl-terminal amino acids, respectively. The difference in observed electrophoretic mobilities between mutants and the full-length rhIL-11-thioredoxin fusion correlated well with the expected molecular masses (Fig. 5). The expression level and solubility of the Delta4 mutant was comparable to that of the wild type rhIL-11, suggesting that removal of 4 residues did not adversely affect the global folding or stability of the protein. Deletion of 8 or more residues had detrimental effects and yielded either an insoluble protein (Delta8) or resulted in substantially decreased protein expression levels (Delta12, Delta16, and Delta20, Fig. 5). rhIL-11 lacking four carboxyl-terminal residues had 25-fold lower bioactivity in the T10 assay (Table 1), whereas elimination of 8 or more residues completely abolished activity. These results suggest that the carboxyl-terminal portion of the rhIL-11 molecule is indispensable for in vitro function, as is observed for other cytokines. Structural studies on the inactive mIL-6 mutant lacking the last 5 residues demonstrated small but significant alterations in regions of the molecule distant from the deletion site, indicating that the carboxyl-terminal tail is also important for proper folding of this cytokine(40) . It cannot be excluded that the loss of activity observed upon deletion of the last 4 residues of rhIL-11 reflects a local structural change in a functionally critical region of the protein. Larger deletions might have resulted in even more pronounced structural alterations, as suggested by the progressive decrease in expression levels and formation of inclusion bodies. For that reason, the functional importance of the carboxyl terminus was further addressed by alanine scanning mutagenesis, in which individual residues in helix D are sequentially replaced by alanine. Results of that study will be presented elsewhere. (^2)


Figure 5: SDS-PAGE of the carboxyl-terminal deletion mutants of rhIL-11, expressed as fusion proteins with E. coli thioredoxin. Proteins present in whole cell lysates (lanes 1, 2, 4, 6, 8, 10, and 12) and soluble cellular fractions (lanes 3, 5, 7, 11, and 13) were analyzed on a 10% SDS-polyacrylamide gel according to Laemmli (29) and stained with Coomassie Blue. Lane 1, host E. coli strain; lanes 2 and 3, full-length rhIL-11 control; lanes 4 and 5, Delta4 mutant; lanes 6 and 7, Delta8 mutant; lanes 8 and 9, Delta12 mutant; lanes 10 and 11, Delta16 mutant; lanes 12 and 13, Delta20 mutant. Positions of fusion proteins are identified by asterisks.



Molecular Modeling

To gain a better understanding of rhIL-11 structure-function relationships, a model of its three-dimensional structure has been constructed. The secondary structure elements in rhIL-11 were established from its amino acid sequence based on the predictive algorithms of Chou and Fasman (33) and Garnier et al.(34) . More than 50% of the residues in rhIL-11 could be assigned to an alpha-helical structure, in good agreement with the results of CD studies (see Fig. 3). Four alpha-helices, connected by peptide segments of variable length, could be differentiated in the proposed structure. The majority of connecting loops lack regular secondary structure, except for the AB loop, which contains a short alpha-helical segment. Such an arrangement of helices and connecting segments is typical of a four-helix bundle topology, a motif which is characteristic of hematopoietic cytokines (Fig. 6A). It is remarkable that rhIL-11 fits into this structural framework, since it contains no cysteines, which in other cytokines form structural elements stabilizing the helical bundle (13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25) , and has unusually high proline content (12%), inconsistent with helical structure. Hydrophobic amino acid side chains, which are found at the interface formed by the alpha-helices in the proposed rhIL-11 bundle (Fig. 6B), could provide the structural stability which compensates for the lack of disulfide bonds. Additionally, a preference for negatively charged side chains in the first helical turn (e.g. Glu and Glu) and positively charged residues in the last turn (e.g. Arg, Arg, Arg, and Lys) may participate in stabilization of the rhIL-11 structure by neutralizing the positive and the negative poles, respectively, of helix dipoles(41) . Smaller clusters of non-polar residues, which are found on the outer face of helices B and D, could provide interaction surfaces to hold connecting segments to the four-helix bundle. A noteworthy feature of the structure is a series of positively charged arginine residues on the face of helix C, which, based on this model, are solvent exposed. Basic amino acids have been noted in the corresponding location on helix C of rhIL-4 and such a ``positively charged wall'' was postulated to be important for the interaction of IL-4 with its receptor(19) .


Figure 6: Proposed four-helix bundle structure of rhIL-11. A, topological diagram. Helical regions are represented by rectangles and are drawn approximately to scale. Arrows indicate amino- to carboxyl-terminal orientation of alpha-helices. The termini of the molecule are marked by the N and C. B, helical wheel projections of 18 residues from each of the alpha-helices expected to form the protein core. Amphipathic character of helices is shown. Hydrophobic faces (crescent-shaped shaded areas) consist mainly of leucine residues, hydrophilic faces contain all charged residues (shaded circles).



The overall topology of rhIL-11, including the length and position of helical segments, is expected to resemble the known structures of hGH (14) and human granulocyte-colony stimulating factor(21) , which are similar in size. This suggestion is further strengthened by the finding that exon/intron boundaries in the genomic structure of hIL-11 (42) mimic those found for hGH (43) and human granulocyte-colony stimulating factor(44) . The IL-6 gene has a similar organization of exons and introns (45) and most likely belongs to the class of four-helix bundle cytokines(26, 27) . It has been suggested that IL-6 and IL-11 represent a distinct subgroup within the family of hematopoietic cytokines(11) .

Preservation of the four-helix bundle topology among cytokines is thought to be important for their interaction with specific cytokine receptors, which are expressed on the cell surface. From our chemical modification and mutagenesis experiments it appears that the rhIL-11 molecule contains two functionally critical regions, which have topology similar to receptor binding sites on hGH(14) . Thus, Met in the AB loop and the carboxyl-terminal residues would contribute to the primary receptor binding site, corresponding to site I of hGH, whereas Lys in loop AB and Lys in loop BC could be involved in site II. In addition to Lys, positively charged arginine residues, which are found on the exposed face of helix C, may play a role in receptor binding to site II. Whether these residues are indeed making contacts with the IL-11 receptor or gp130 subunit will have to be determined.

IL-11 from monkey (32) and mouse (^3)demonstrate considerable identity with the human sequence, especially in the putative alpha-helical regions of the molecule. Met, Lys, and Lys are invariant among these species. All these proteins have full cross-species reactivity indicating that their tertiary structures are similar and other functionally critical residues are probably conserved.

Limited Proteolysis

The proposed model of rhIL-11 structure was probed by limited proteolysis, assuming that the compact alpha-helical bundle would be more resistant to hydrolysis than protruding interconnecting loops and the amino terminus of the molecule. rhIL-11 was digested under native, nondenaturing conditions, using three proteases of different side chain specificities, endoproteinase Asp-N, trypsin, and chymotrypsin. Under limiting conditions, all three proteases selectively recognized only a small subset of potential cleavage sites, indicating the presence of protein domains differing in enzyme accessibility (Fig. 7). Fragmentation of the molecule occurred mostly in the postulated loop regions of the molecule and at the amino and carboxyl termini of putative alpha-helices. In agreement with the chemical modification data, peptide bonds adjacent to Met (but not Met), Lys, and Lys were selectively accessible to protease action. In contrast, regions of alpha-helix were resistant to hydrolysis under the conditions used, even though they contain multiple cleavage sites for all the enzymes, except for a single accessible peptide bond in both helix D and helix A (Fig. 7). This pattern of cleavage is consistent with the proposed four-helix bundle structure of rhIL-11. Verification of this model, however, will require direct IL-11 structure determination, either by x-ray crystallography or by multidimensional NMR.


Figure 7: Limited proteolysis of rhIL-11 with endoproteinase Asp-N, trypsin, and chymotrypsin. Location of protease-sensitive sites in the proposed four-helix bundle of rhIL-11.




FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Genetics Institute, One Burtt Rd., Andover, MA 01810.

(^1)
The abbreviations used are: IL, interleukin; rhIL, recombinant human interleukin; MOPS, 3-(N-morpholino)propanesulfonic acid; MES, 2-(N-morpholino)ethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; FAB/MS, fast atom bombardment mass spectrometry; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; hGH, human growth hormone; HPLC, high performance liquid chromatography.

(^2)
Czupryn, M. J., McCoy, J. M., and Scoble, H. A. (1994) Ann. N. Y. Acad. Sci., in press.

(^3)
J. C. Morris and C. R. Wood, unpublished results.


ACKNOWLEDGEMENTS

We wish to thank Paul Schendel for helpful discussions and comments on the manuscript, Michael Brenner for amino-terminal amino acid sequencing, Wen Yu and Steve Martin for mass spectral analysis of samples, Frann Bennett and Sara Yankelev for sample bioassays, Hemchand Sookdeo and Jennifer Dube for DNA synthesis, Neil Schauer, Steve Vicik, and Jenifer Thomas for purification of M58A mutant protein, John Steckert for CD spectroscopy, and Lisa Housianitis for help in the preparation of the figures.


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