From the Biomolecular Research Institute, 343 Royal
Parade, Parkville 3052, Australia and the § Cooperative
Research Centre for Cellular Growth Factors and the
Walter and
Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital,
Melbourne 3050, Australia
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ABSTRACT |
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The solution structure of a murine-human chimera
of leukemia inhibitory factor (LIF), a 180-residue cytokine with a
molecular mass of 20 kDa, has been determined using multidimensional
heteronuclear NMR techniques. The protein contains four -helices,
the relative orientations of which are well defined on the basis of
long-range interhelical nuclear Overhauser effects. The helices are
arranged in an up-up-down-down orientation, as found in other
four-helix bundle cytokines, and the overall topology of the chimera is
similar to that of the crystal structure of murine LIF (Robinson,
R. C., Grey, L. M., Staunton, D., Vankelecom, H. Vernallis,
A. B., Moreau, J. F., Stuart, D. I., Heath, J. K.,
and Jones, E. Y. (1994) Cell 77, 1101-1116).
Differences between the structures are evident in the N-terminal
region, where the peptide bond preceding Pro17 has a
trans-conformation in solution but a
cis-conformation in the crystal, and in the small
antiparallel
-sheet encompassing residues in the N terminus and the
CD loop in the crystal structure, which is not apparent in solution.
There are also minor differences in the extent of the helices. Other
than at the N terminus, the main difference between the two structures
occurs at the C-terminal end of the CD loop. As this loop is close to a
receptor-binding site on LIF that makes a major contribution to high
affinity binding to the LIF receptor
-chain, these differences
between the solution and crystal structures should be taken into
account in structural models of LIF receptor interactions.
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INTRODUCTION |
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Leukemia inhibitory factor (LIF)1 is a glycoprotein identified and purified on the basis of its ability to induce differentiation in murine myeloid leukemic M1 cells (1). LIF is produced, however, in a diverse array of cell types and is a highly pleiotropic cytokine, exerting a range of actions on a number of cell types, including hepatocytes; adipocytes; megakaryocytes; neuronal, muscle, and embryonic stem cells; and osteoblasts (2). These actions include the induction of cell proliferation (in myoblasts and megakaryocytes), the induction (in M1 cells) or suppression (in embryonic stem cells) of differentiation, and the induction of mature cell function (in neurons, adipocytes, and hepatocytes) (2, 3).
Although it has multiple activities, LIF appears to be absolutely
necessary only for embryo implantation. Male mice lacking the gene for
LIF are apparently normal, whereas the corresponding female mice are
infertile, and the deficiency can be corrected by injection of purified
LIF (4). In contrast, mice lacking the LIF receptor have multiple
placental, skeletal, neural, and metabolic disorders, which lead to
perinatal death (5). The explanation for these observations, at least
in part, is that several other cytokines share components of the LIF
receptor, the functional form of which consists of the LIFR -chain
and a common
-chain, gp130 (3). The
-chain is shared with
interleukin (IL)-6 and IL-11, and both chains form part of the receptor
complexes for oncostatin M, ciliary neurotrophic factor (CNTF), and
cardiotrophin-1.
With its range of biological functions and activity against a variety of target cells, LIF also has potential therapeutic applications. Its ability to induce the growth and differentiation of peripheral and central nerve cells indicates a potential use in the treatment of peripheral nerve damage (6) and motor neuron disease. Furthermore, the receptor subunit gp130, which is shared among the IL-6 family of cytokines, is a potential target for antagonists that could be used in treating certain cancers and autoimmune diseases associated with overproduction of these cytokines (7). A knowledge of the structures of the cytokines and their receptor complexes will open the way for the design of cytokine antagonists or agonists directed toward specific tissues.
Despite the low sequence identity among the four-helix bundle cytokines
(17-24%) (8), there are common features of their three-dimensional
structures. On the basis of their sequences and tertiary structures,
they have been divided into two classes (9-11), which share a common
motif consisting of four helices (referred to as the A, B, C, and D
helices) arranged in an antiparallel up-up-down-down fashion, with long
loops between the A and B helices and the C and D helices. The
short-chain molecules are distinguished from their long-chain
counterparts by the lengths of their helices, which span only 15 or so
residues compared with ~25 in the long-chain class, and by the
secondary structure elements in the long AB and CD loops, with the
short-chain molecules typically having a small -sheet and the
long-chain molecules having
-helices.
We have undertaken an investigation by NMR spectroscopy of the solution structure of LIF. Mature LIF has three disulfide bonds and is heavily N-glycosylated, with an apparent molecular mass range of 32-62 kDa in the glycosylated state, but only 20 kDa in the non-glycosylated state (12). Samples for NMR were obtained from Escherichia coli, where the protein is not glycosylated, but it has been shown that glycosylation is not important for activity (2, 13). The solution structure was determined for MH35-LIF, a murine-human chimera of LIF consisting of residues 1-47 and 83-180 of mLIF and residues 48-82 of hLIF, except for residues 107, 112, 113, 155, and 158, which are from hLIF (Fig. 1). hLIF binds both the human and murine receptors with high affinity, whereas mLIF binds strongly only to the murine receptor (14, 15). Although MH35-LIF has a greater sequence similarity to murine LIF than to human LIF, it has essentially the same biological activity as hLIF (15), and thus, it was of interest to determine its structure to characterize changes in the receptor-binding domains of the protein. MH35-LIF also expressed at high levels in E. coli grown on minimal medium and could be labeled efficiently (16).
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EXPERIMENTAL PROCEDURES |
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NMR Spectroscopy-- The preparation of 13C- and 15N-labeled MH35-LIF and the chemical shift assignments and secondary structure of MH35-LIF have been reported previously (17). NMR data were acquired on samples at pH 4.4 and 40 °C.
A two-dimensional NOESY spectrum was acquired on unlabeled MH35-LIF with a 150-ms mixing time, and the following spectra were acquired on uniformly 15N-labeled MH35-LIF: three-dimensional 1H,15N NOESY-HSQC (18) with a 150-ms mixing time to obtain distance constraints, HNHA (19) or HMQC-J (20) to determine NH-CStructural Constraints--
Approximate interproton distances
were derived from two-dimensional NOESY and three-dimensional
13C- and 15N-edited NOESY spectra. Peak
intensities were classified as strong, medium, weak, or very weak on
the basis of contour levels and translated into upper distance bounds
of 3.0, 4.0, 5.0, and 6.0 Å, respectively, and corrections for
pseudoatoms were added. A total of 1599 interresidue and 900 intraresidue constraints were used in the final structure calculations.
Hydrogen bond restraints were employed for backbone amide protons where
there was a slowly exchanging amide proton (27), a low
NH-CH coupling constant, and medium-range NOEs
consistent with the presence of an
-helix (28). Each hydrogen bond
was constrained by an NH-O distance restraint of 2.3 Å and an N-O
distance of 3.3 Å, yielding a total of 156 constraints in the
structure calculations. The disulfide bonds were constrained by setting
ranges of 2.0-2.1 and 3.0-3.1 Å, respectively, for the
Si-Sj and Si-C
j
distances of each bond.
Structure Calculations-- Initial structures were generated with the torsion angle dynamics program DYANA (29). Several rounds of structure calculation were carried out using DYANA to resolve violated distance constraints and to determine possible assignments for ambiguous NOE cross-peaks. This process was repeated until all the distance and angle restraints produced a set of structures that had no NOE distance violations >0.3 Å or dihedral angle violations >5°. Once the final set of restraints had been obtained, a new family of structures was generated using DYANA; the 50 structures with the lowest penalty functions were selected from a calculation of 1000 structures, and these were refined in X-PLOR (30) using dynamical simulated annealing (31) and energy minimization, as described previously (28), but without neutralization of charged side chains. The 20 best structures, on the basis of their stereochemical energies, were chosen for structural analysis.
Structures were analyzed using MOLMOL (32) and PROCHECK_NMR (33). Hydrogen bonds were identified in MOLMOL using a maximum C-N distance of 2.4 Å and a maximum angular deviation of 35° from linearity. Structural figures were generated using MOLMOL. ![]() |
RESULTS |
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Structure Determination-- Structural restraints were determined from a combination of two- and three-dimensional double- and triple-resonance experiments using both 15N- and 13C,15N-labeled samples as well as unlabeled protein. A representative region of a three-dimensional 13C NOESY-HSQC spectrum, from which numerous distance restraints were obtained, is shown in Fig. 2. There were very few constraints for the first nine residues, which are apparently disordered in solution.
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Structure of MH35-LIF-- The overall topology is a four-helix bundle, with the helices arranged in an up-up-down-down left-handed fashion. The N-terminal region is distinguished by its lack of long-range order, with the first nine residues being unstructured and residues 10-22 being poorly ordered. The A helix begins at residue 24 and contains a marked kink in the vicinity of residues 34-36. This kink, which was also observed in the crystal structure of mLIF (34), is reflected in the backbone amide exchange behavior of the amides (27)2 and the chemical shifts for residues in this region (17). The first of the two long interhelical loops, the AB loop, connects the C terminus of the A helix with the N terminus of the B helix. The AB loop contains some elements of secondary structure near its N terminus, with two irregular turns preceding a short helix between residues 55 and 60. This loop is well packed against the helical bundle, with long-range NOEs observed between residues in the loop (Leu56, Leu59, and Cys60) and residues in the A and D helices. There is a very short, well ordered loop linking the B and C helices. The C and D helices are connected by the second long loop. In contrast to the AB loop, the CD loop has no regular secondary structure and is less ordered overall, as judged by the angular order parameters and RMSD values over these residues compared with the AB loop (Fig. 3).
Comparison with Crystal Structure of mLIF--
Fig. 3 shows the
residue by residue RMSD from the crystal structure of mLIF (34). The
overall RMSD when the two structures are aligned over the backbone of
the common helical bundle residues is 1.55 Å, indicating good
agreement. Comparison of the uncertainty in the positions of the atoms
as measured by crystallographic B factors in mLIF (Fig. 3G)
with the backbone RMSDs of the solution structure (Fig. 3D)
shows that they follow a similar pattern, emphasizing the similarity
between the two structures. The lengths of the four main helices in the
solution structure differ slightly from those of the x-ray structure,
with the limits for the helices (according to PROCHECK_NMR) being
24-45, 76-104, 108-133, and 154-176 for the A, B, C, and D helices,
respectively, compared with 22-48, 76-104, 109-135, and 155-177 in
the crystal structure. The short helix involving residues 55-60 is
also present in the crystal structure. Analysis of the crystal
structure using PROCHECK shows that there is a short antiparallel
-sheet involving residues 10-12 in the N-terminal tail and residues
140-142 in the CD loop, but this is not present in solution.
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DISCUSSION |
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Comparison with Proteins of Related Folds-- A number of structures have been determined for four-helix bundle cytokines or related molecules by either NMR or x-ray crystallography. Six of these, G-CSF, growth hormone, CNTF, IL-6, leptin, and LIF itself, are in the "long-chain" class of cytokine. The sequence identity among these molecules is low, but they share common structural motifs and similar receptor systems. In particular, LIF, IL-6, and CNTF all share the signal-transducing protein gp130, and this is reflected in at least one common receptor-binding site among this group of cytokines.
The solution structure of MH35-LIF is in good agreement with that of mLIF determined by x-ray crystallography (34). As well as sharing the overall antiparallel four-helix bundle motif, structural details similar to those determined for MH35-LIF have been observed for G-CSF (35), CNTF (36), IL-6 (37, 38), and human leptin (39). The A helix has a kink approximately half way along its length, as also found for CNTF, IL-6, and leptin in a similar although not identical position. Although there is no three-dimensional structure available for oncostatin M, differences from chemical shift patterns expected for a purelyReceptor Interactions--
The receptor complex for LIF is
believed to involve a single ligand molecule in association with gp130
and LIFR, and this ternary complex is responsible for signal
transduction. A partial alanine scan of hLIF (41) suggested that there
were three distinct receptor-binding sites on the surface of LIF,
designated sites I-III. Similar sites have been identified on the
surfaces of CNTF (36, 42) and IL-6 (37, 38). Site I, at the C terminus of the D helix, confers specificity of the ligands IL-6, CNTF, and LIF
for their respective receptor -chains, whereas site II, at the A-C
helical interface, interacts with the signal-transducing protein gp130.
Site III, at the N terminus of the D helix and the N-terminal end of
the C helix, interacts either with LIFR in the cases of CNTF and LIF or
with a second gp130 molecule in the case of IL-6. The locations of
these sites on the structure of MH35-LIF are shown in Fig.
5A.
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Post-translational Modifications--
LIF is
N-glycosylated on Asn residues 9, 34, 63, 73, 96, and 116 when expressed in Chinese hamster ovary cells (44). Structurally, these
sites on LIF do not form a single contiguous surface, but are
distributed over the surface (Fig. 5C). The sites are
located in the unstructured N-terminal tail (Asn9), the
middle of the A helix near the kink (Asn34), the AB loop
(Asn63 and Asn73), the C-terminal end of the B
helix (Asn96), and the N-terminal end of the C helix
(Asn116). As the degree of glycosylation does not appear to
affect the interaction of LIF with its receptor, the carbohydrate
moieties must not interfere with receptor interaction. Three of the
sites (Asn residues 63, 96, and 116) encircle site III, but are
directed away from it; Asn96 and Asn63 are ~9
Å away from site III. Asn34 lies between sites I and II,
but the location of these sites on opposite faces of LIF accounts for
the lack of effect of glycosylation on binding to either gp130 or the
LIFR -chain.
Histidine Environments-- Previously, we determined the imidazolium pKa values for the six His residues of MH35-LIF using two-dimensional 1H NMR (16). Three of these are in the range 6.0-6.4, but the other three are perturbed, and the structure accounts satisfactorily for these observed effects. His71 is close to Glu82, which is likely to be responsible for the low pKa inflection in the titration curve for His71 and the elevated imidazolium pKa (>7.5). His112 is very close to the side chain of Lys102 and is partially buried, which both would contribute to its low pKa (4.1), and His150, with a pKa of 5.4, is also partially buried.
Small Molecule Design--
One approach to the development of
small molecule agonists or antagonists of cytokines is the screening of
oligopeptide libraries displayed on bacteriophage surface proteins. An
alternative strategy is the de novo design of mimetics of
individual receptor sites on the cytokine surface, although this
approach is probably more suited to the generation of an antagonist
than an agonist. The IL-6 family of cytokines has similar
three-dimensional structures and three topologically conserved binding
sites, and there are even greater similarities among those members of
the family that bind to the LIFR -chain, e.g. the common
D1 motif in site III. A small molecule that mimicked site III of the
LIF family of cytokines by binding tightly to the LIFR
-chain might
have a wider spectrum of activities than desirable therapeutically, as
it would inhibit the actions of all members of the LIF family (41). An
antagonist that bound tightly to LIF itself would overcome this
problem. The detailed knowledge now available of the structure of LIF
and the residues contributing to the three receptor-binding sites on
its surface also opens the way for a structure-based approach to the
development of low molecular mass analogues.
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ACKNOWLEDGEMENTS |
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We thank John MacFarlane for assistance with computing and Peter Güntert for provision of the program DYANA-1.0.
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FOOTNOTES |
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* This work was supported by the Cooperative Research Center for Cellular Growth Factors.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The structures and the NMR restraints on which they were based (codes 1a7m and 1a7mmr) have been submitted to the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
¶ Present address: Dept. of Biophysics and Physical Biochemistry, Regensburg University, 93053 Regensburg, Germany.
** To whom correspondence should be addressed. Fax: 61-3-9903-9655; E-mail: ray.norton{at}molsci.csiro.au.
1 The abbreviations used are: LIF, leukemia inhibitory factor; mLIF, murine LIF; hLIF, human LIF; LIFR, LIF receptor; IL, interleukin; CNTF, ciliary neurotrophic factor; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; HSQC, heteronuclear single-quantum coherence spectroscopy; RMSD, root mean square deviation; G-CSF, granulocyte colony-stimulating factor.
2 S. Yao, manuscript in preparation.
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REFERENCES |
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