(Received for publication, August 15, 1994; and in revised form, November 4, 1994)
From the
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.
-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.
Interleukin-11 (IL-11) ()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 -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.
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.
For cation exchange HPLC, a Bakerbond wide-bore CBX column
was used (0.46 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.
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.
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.
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%
-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.
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.
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, 4 mutant; lanes 6 and 7,
8 mutant; lanes 8 and 9,
12 mutant; lanes 10 and 11,
16 mutant; lanes 12 and 13,
20 mutant. Positions of fusion
proteins are identified by asterisks.
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 -helices. The termini of the molecule are marked by the N and C. B, helical wheel projections of 18
residues from each of the
-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 ()demonstrate considerable identity
with the human sequence, especially in the putative
-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.
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.