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Research Article 863
Crystal structure and functional dissection of the cytostatic cytokine oncostatin M Marc C Deller1,2†#, Keith R Hudson2‡#, Shinji Ikemizu1, Jerónimo Bravo1§, E Yvonne Jones1* and John K Heath2
Background: The cytokine oncostatin M (OSM) inhibits growth of Addresses: 1Cancer Research Campaign Receptor certain tumour-derived cells, induces proliferation in other cell types Structure Group, Division of Structural Biology, The Wellcome Trust Centre for Human Genetics, (e.g. haemangioblasts) and is a mediator of inflammatory responses. Its University of Oxford, Roosevelt Drive, Oxford OX3 mechanism of action is via specific binding to gp130 and either the leukaemia 7BN, UK and 2Cancer Research Campaign Growth inhibitory factor receptor (LIFR) or oncostatin M receptor (OSMR) systems Factors Group, School of Biochemistry, University of at the cell surface to form an active signalling complex. Birmingham, Edgbaston, Birmingham B15 2TT, UK. Present addresses: †Yale University School of Results: We report here the crystal structure of human oncostatin M (hOSM) Medicine, Department of Pharmacology, Sterling along with mutagenesis data which map the receptor-binding epitopes of the Hall of Medicine, PO Box 208066, New Haven, molecule. The structure was determined to a resolution of 2.2 Å and conforms CT 06520-8066, USA, ‡Genesis Research and to the haematopoietin cytokine up-up-down-down four-helix bundle topology. Development, 1 Fox Street, Parnell, Auckland, New Zealand and §Laboratory of Molecular Biology, The site 2 epitope, responsible for gp130 binding, is centred around Gly120 Medical Research Council Centre, Hills Road, which forms a ‘dimple’ on the surface of the molecule located on helices A and Cambridge CB2 2QH, UK. C. The site 3 motif, responsible for LIFR and OSMR binding, consists of a protruding Phe160/Lys163 pair located at the start of helix D. *Corresponding author. E-mail: [email protected]
Conclusions: The data presented allow functional dissection of the #These authors contributed equally to this work. receptor-binding interfaces to atomic resolution. Modelling suggests that the gp130 residue Phe169 packs into the site 2 dimple in an analogous fashion Key words: four-helix bundle cytokine, gp130 binding, oncostatin M, structure–function to structurally equivalent residues at the growth hormone–growth hormone receptor interface, implying that certain key features may underlie recognition Received: 26 April 2000 across the whole cytokine/receptor superfamily. Conversely, detailed Revisions requested: 7 June 2000 comparison of the available structures suggests that variations on a common Revisions received: 22 June 2000 Accepted: 28 June 2000 theme dictate the specificity of receptor–ligand interactions within the gp130 family of cytokines. Published: 20 July 2000
Structure 2000, 8:863–874
0969-2126/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved.
Introduction hOSM is a member of the family of gp130 cytokines that Human oncostatin M (hOSM) is a secreted 252 amino acid are characterised by the use of the shared transmembrane polypeptide cytokine that was originally isolated by virtue transducing receptor gp130 in their mechanism of action of its ability to suppress the proliferation of human [11]. hOSM is therefore functionally related to leukemia melanoma cells in vitro [1]. Subsequent biological investiga- inhibitory factor (LIF), interleukin (IL)-6 and IL-11, car- tions have revealed that hOSM and mouse OSM (mOSM) diotrophin-1 (CT-1) and ciliary neurotrophic factor exhibit a diversity of effects on different target cell types. (CNTF). A key feature of the gp130 cytokines is that These include the ability to inhibit the multiplication of a intracellular signalling results from the ligand-mediated variety of different tumour-derived cell lines (reviewed in formation of oligomeric receptor complexes which, [2]) as well as inducing the proliferation of certain cells depending upon the identity of the ligand, can differ in including Kaposi sarcoma-derived spindle cells [3] and hae- both composition and stoichiometry [12]. In all cases, mangioblasts derived from the aorta–gonad–mesonephros however, receptor complexes include one or more mol- region of the developing embryo [4]. hOSM, released from ecules of the shared transducer gp130 [13]. gp130 macrophages and neutrophils [5], has also been implicated cytokines can therefore exhibit both unique and shared as an important mediator of pro-inflammatory responses in biological activities in vivo and in vitro depending upon the joint destruction [6] and central nervous system inflamma- composition of the receptor signalling complex that is tion and neurodegeneration [7,8] via, inter alia, the induc- formed [11]. hOSM signals via formation of an active tion of acute- phase protein gene expression [9,10]. receptor signalling complex that comprises gp130 and one 864 Structure 2000, Vol 8 No 8
of two partner receptors: either the LIF receptor (LIFR) cases, receptor specificity is controlled by subtle structural [14] or the structurally related OSM-specific receptor, changes to a common recognition framework. OSMR [15]. mOSM [16] differs from hOSM in that it only signals via association with gp130 and the murine OSMR Results [17,18]; this means that there is a significant divergence in Expression and structure determination action between mOSM and hOSM and that the LIFR A soluble 21.4 kDa fragment of hOSM was expressed in recognition elements of OSM are not conserved. This Escherichia coli as a glutathione-S-transferase (GST) fusion finding also indicates that the unique biological activities protein. The resulting purified protein consists of residues of hOSM, such as cytostatic effects on tumour cells, are 1–187 of hOSM and a further four residues at the N termi- mediated via association with OSMR. Those activities of nus corresponding to the 3C protease site linker (GPGS). hOSM that are shared with other cytokines, such as LIF or Ba/F3 cell-survival assays and receptor-binding assays con- CNTF, may reflect a common association with the LIFR. firmed that this recombinant form binds gp130 with an affinity equivalent to wild-type (WT) hOSM. Knowledge of the structural basis of receptor recognition by gp130 cytokines is a central issue in understanding both Crystallisation trials produced Bragg diffraction quality the specificity of their biological functions and developing crystals belonging to the orthogonal space group P212121, methods for therapeutic intervention in gp130-mediated with unit-cell dimensions a = 35.8 Å, b = 53.1 Å, c = 106.8 Å. signalling. The crystal structures of three gp130 cytokines, The structure was determined from a single ethylmer- LIF [19], CNTF [20] and IL-6 [21], have been previously curyphosphate (EMP) derivative using the multiple described. These reveal that all three cytokines share a anomalous dispersion (MAD) phasing technique with common three-dimensional fold, the four-helix bundle X-ray diffraction data collected on beam line BM14 of the [22], which comprises four α helices ranging from 15 to 22 European Synchrotron Radiation Facility (ESRF). The amino acids in length (termed A, B, C and D) and linked structure has been refined to a crystallographic R factor of by polypeptide loops. Detailed structure–function studies 20.5% (Rfree 26.1%) for all data between 20.0 and 2.2 Å, of human LIF, which is most closely related to hOSM in with stereochemistry typified by root mean squared devia- sequence and receptor repertoire, have defined two recep- tions (rmsd) in bond lengths of 0.015 Å and in bond angles tor recognition epitopes: sites 2 and 3. Site 2 (so-termed by of 1.8°. Crystallographic statistics are reported in Tables 1 analogy to the receptor-binding sites in growth hormone and 2. Residues 1–3 and 135–155 appear to be disordered [23]), is required for recognition of gp130 and has been and are not included in the final model. With the excep- narrowed down to a cluster of candidate amino acid tion of these regions, and a flexible loop between residues residues located in helices A and C [24]. Site 3 is required Glu90 and Lys110, the electron density is good through- for recognition of LIFR and is dominated by a pair of con- out the course of the mainchain (a representative portion served amino acid residues located at the N-terminal tip of of electron density is shown in Figure 1a). Residue num- helix D [24]. The combined activity of these two recogni- bering is that of the human native mature protein tion epitopes leads to the formation of a trimeric, LIF- (GenBank accession number M27286). mediated, high-affinity signalling complex [24]. Structure description Structural comparisons of ligands with differing recognition hOSM is a compact barrel-shaped molecule with dimen- properties present a valuable opportunity to analyse the sions of approximately 20 Å × 27 Å × 56 Å that conforms molecular determinants that dictate receptor specificity. to the up-up-down-down four-helix bundle topology Here, we report the determination of the three-dimensional (Figure 1b,c). The basic fold consists of the four main structure of hOSM by crystallographic techniques and con- helical regions (helix A, residues 10–37; helix B, residues comitant functional dissection by mutagenesis. Because 67–90; helix C, residues 105–131; helix D, residues hOSM binds to gp130 with a higher affinity than LIF [24], 159–185) connected by two long overhand loops (AB loop, this allows structure–function analysis of the site 2 gp130 residues 38–66; CD loop, residues 130–158) and one short recognition epitope at the resolution of individual amino loop (BC loop, residues 91–104). The overall arrangement acid residues. Combining these findings with the high-reso- may be considered as two pairs of antiparallel helices that lution crystal structure of the complementary ligand-recog- pack together, with A–D forming one pair and B–C nition epitope located in the cytokine receptor homology forming the other. domain (CHD) of gp130 [25] results, for the first time, in a definition of the structure of the interface between hOSM Both helices A and C exhibit breaks in the classical hydro- and gp130. In addition a detailed comparison of the three- gen-bonding pattern, with substitute hydrogen bonds dimensional structure of the site 3 recognition epitope of formed to tightly bound water molecules. In helix C this hOSM and hLIF also offers insights into the molecular results in a slight kink (between residues Gln112 and basis by which related ligands exhibit different receptor Pro116). The kink in helix A is more pronounced and is recognition properties. These findings reveal that, in both caused by a break in the helical conformation, as residues Research Article Crystal structure of oncostatin M Deller et al. 865
Table 1
Data collection statistics.
λ λ λ Native EMP 1 EMP 2 EMP 3
Wavelength (Å) 0.979273 1.00914 0.826516 1.00669 Unit cell a,b,c (Å) 35.8, 53.1, 106.8 35.9, 53.3, 106.7 α = β = γ (°) 90 90 Resolution range (Å) 25–2.2 25–2.6 Total reflections 43,901 24,040 26,223 24,159 Unique reflections 10,513 11,650 12,069 11,691 Redundancy 4.2 2.1 2.2 2.1 Completeness (%) 96.5 (98.2) 96.4 (63.1) 99.9 (100) 96.8 (66.7) 13.2 (4.3) 12.1 (2.9) 12.5 (4.7) 12.3 (3.3)
Rmerge (%) 8.3 (37.6) 4.6 (18.7) 4.6 (16.4) 4.3 (17.3) λ Σ Σ EMP denotes MAD data collected from EMP-derivatised hOSM. Rmerge = |I–|/ . Values in parentheses correspond to the highest resolution shell (2.66–2.60 Å for EMP, 2.25–2.20 Å for native).
Gln25 and Leu30 adopt a hydrogen-bonded turn confor- LIF [19,27]. Structural superimpositions with these and mation, with four tightly bound water molecules substitut- other four-helix bundle cytokines show that the gp130 ing for the classical hydrogen-bonding pattern (Figure 1a). cytokines form a distinct family both structurally and
Residues Thr27–Ile37 of helix A adopt a 310 helix confor- functionally. Within the gp130 cytokine family hOSM has mation. The curved nature of helices A and C facilitates the greatest structural similarity to LIF (rmsd of 2.1 Å close packing of the A–D and B–C helix pairs, so promot- over 145 pairs of structurally equivalent Cα atoms). The ing an extensive solvent-inaccessible core. more distant structural similarity to the four-helix bundles of the growth hormone family is at its best for superimpo- The core of the protein is composed of two groups of aro- sition of hOSM and erythropoietin (EPO; [28]) (rmsd of matic stacking interactions in which Phe56, Tyr173, 2.8 Å for 120 pairs of structurally equivalent Cα atoms). Phe169 and Phe176 form one group and Phe70, Phe185 and Trp187 form another. This distribution emphasizes The addition of hOSM to the gp130 cytokine structural the hydrophobic nature of helix D that donates all of the database highlights several features of potential functional aromatics to these groupings, with the exception of Phe56 (AB loop) and Phe70 (B helix). Table 2
The N-terminal loop preceding helix A (Gly4–Glu9) is Refinement statistics. tethered to the C terminus of helix C by one of the two disulphide bridges (Cys6 and Cys127). The second disul- Resolution range (Å) 20–2.2 (2.28–2.20) R (%) 20.5 (27.1) phide bridge, between Cys49 and Cys167, anchors the cryst Rfree (%) 26.1 (33.6) start of the AB loop to the N-terminal region of helix D. Completeness The AB loop itself consists of two separate α-helical working set (%) 86.1 (81.0) regions running from Pro43 to Arg46 and Glu59 to Gly64. test set (%) 9.9 (8.6) Residues between these two helical regions adopt an No. of reflections (F > 0) working set 9410 (870) extended conformation and pack closely against helix D. test set 1082 (92) By contrast, the second two loops (BC and CD) are less No. of non-hydrogen atoms closely associated with the core structure. The BC loop protein 1319 protrudes above the four-helix bundle and exhibits higher water 116 than average B factors. Despite this relative mobility it Rmsd from ideality bond lengths (Å) 0.0147 does, however, contain a high percentage of regular sec- bond angles (Å) 1.7997 ondary structure with a 310 helix running between residues dihedrals (°) 19.8882 Ala95 and Asp97 followed by an α helix up to residue improper (°) 1.0381 Ser101. The long CD loop appears to be highly mobile Average B factors mainchain (Å2) 27.1 and could not be modelled as a single conformation. sidechain (Å2) 32.4 water (Å2) 38.4 Structural comparison with other helical cytokines all atoms (range) (Å2) 30.5 (6.3–84.9) hOSM is the fourth member of the gp130 cytokine family Σ Σ Rcryst = ||Fobs|–|Fcalc||/ |Fobs|. Rfree is as for Rcryst but calculated using a to be analysed structurally; crystal and/or solution struc- 10% test set of reflections excluded from the refinement. Values in tures have been reported for IL-6 [21,26], CNTF [20] and parentheses correspond to the highest resolution shell (2.28–2.20 Å). 866 Structure 2000, Vol 8 No 8
Figure 1
The structure of hOSM. (a) Refined α coordinates and 2|Fo|–|Fc| calc electron-density map of hOSM contoured at 1σ showing density for the kinked region of helix A centred on Thr27. CNTF and LIF also have kinks owing to disruption of the mainchain hydrogen bonding around residues Thr32 and Ser36, respectively [19,20]. Both these residues bind water molecules through the Oγ1 atom to provide a substitute hydrogen-bond network similar to the pattern observed here for hOSM involving residue Thr27. (b) Ribbon diagram of hOSM coloured from blue at the N terminus through to red at the C terminus. Disulphide bonds are represented as ball-and-stick models with the sulphur atoms highlighted as yellow spheres. The transparent dotted section represents the CD loop as observed in LIF. (c) Stereodiagram of the Cα trace for hOSM.
importance (Figure 2). First, the kinked helix A is as having the dominant role in ligand binding. Comparison revealed as a distinctive feature not just of the gp130 of loop structures between family members appears to cytokines but also specifically of the members of the bear this out as there are few clearly conserved characteris- family of cytokines that bind to LIFR; IL-6 is the sole tics. Each crystal structure, except LIF, indicates highly gp130 cytokine lacking this feature (Figures 1a,2a). The flexible regions in the long interhelix loops; however, kink results in a repositioning of the C-terminal end of these vary in location between the AB loop in CNTF and helix A relative to the ‘top’ of the four-helix bundle, a IL-6, and the CD loop in CNTF and hOSM. Similarly the region carrying the major epitope for LIFR, and OSMR, positioning of disulphides tethering loop regions to the binding (at the start of helix D). This feature is therefore core four-helix bundle is not conserved. It has been noted, suggestive of the positioning of the C-terminal end of however, that the positioning, relative to helix D, of the helix A impacting on LIFR and OSMR binding. Con- N-terminal portion of the AB loop in LIF differs from that versely, the positioning of the N-terminal portion of in the distantly related four-helix bundle cytokines [19]. helix A, which is implicated in gp130 binding, is relatively The current structural comparisons may point to some conserved across all members of the family. functional correlation of this feature with LIFR binding as both LIF and hOSM have a disulphide bridge anchoring In general, functional studies on the gp130 cytokines have an α-helical N-terminal portion of the AB loop to the top implicated the helices, rather than the connecting loops, of helix D (Figure 2a). Research Article Crystal structure of oncostatin M Deller et al. 867
Figure 2
Structural comparisons. (a) From left to right the structures of human OSM, LIF (PDB entry code 1EMR), CNTF (1CNT) and IL-6 (1ALU) are shown in equivalent orientations. Each is represented as a ribbon coloured blue at the N terminus through to red at the C terminus. Helix A (blue) is kinked in OSM, LIF and CNTF whereas helix C (light green/yellow) is only significantly kinked in OSM. Of the interhelical loops, only the relatively short BC loop is well determined in all the structures. This region in hOSM has a five-residue insertion that protrudes from the top of the four-helix bundle. Both LIF and hOSM have a disulphide bridge anchoring an α-helical N-terminal portion of the AB loop to the top of helix D, whereas in IL-6 the loop is anchored to helix B. The AB loop in CNTF lacks any such tether and was thus too mobile to be accurately modelled in the crystal structure. The CD loop has not been fully modelled for hOSM and CNTF, although the orientation of residues preceding this loop suggests a similar route is taken to that of LIF, IL-6 and CNTF. (b) Comparison of surface charge between hOSM, LIF, CNTF and IL-6. Blue denotes positive and red negative; electrostatic potential is contoured at ±8.5 kT using the program GRASP [53]. The orientation is as in (a). (c) Surface charge comparisons are displayed as in (b) but viewed directly onto the top of the four-helix bundle.
Sequence conservation across the gp130 cytokines is rela- (KRH and JKH, unpublished observations) derived using tively low (i.e., typically some 20% sequence identity); the crystal coordinates of human LIF (hLIF; Protein Data therefore very limited conservation of surface character is Bank [PDB] ID 1EMR) as a template and previously pub- to be expected. Figures 2b and 2c illustrate a comparison lished work on the location of receptor-recognition epi- of the surface characteristics in two regions of functional topes in human LIF [24] and CNTF [29]. Only mutation significance; for both regions there is notable conservation of those residues that proved to exhibit significant solvent between three of the four gp130 cytokines. At the puta- exposure (Table 3) in the crystal structure of hOSM are tive gp130-binding region involving helix A (Figure 2b), considered here. CNTF exhibits a significantly different electrostatic surface potential, whereas for the putative LIFR binding Mutant proteins were tested for their ability to inhibit the site 3 on the top of the four-helix bundle (Figure 2c), IL-6 binding of biotin-labelled hOSM to immobilised human is the outlier. gp130 or human LIFR ectodomains in solid-phase binding assays (Figure 3). It was not possible to analyse Site-directed mutagenesis of hOSM binding to hOSMR in this assay format owing to the sig- A panel of alanine scanning mutants of hOSM was gener- nificantly reduced affinity of hOSM for OSMR compared ated in order to identify critical residues involved in recep- to LIFR [18]. Mutant proteins were also tested for their tor recognition. The selection of residues for mutagenesis ability to promote the multiplication of BAF cells trans- was initially on the basis of a structural model of hOSM fected with either human LIFR/human gp130 [24] or 868 Structure 2000, Vol 8 No 8
Table 3
Mutagenesis data for hOSM.
Location/mutant Site RSA (%)* Receptor-binding assays Ba/F3 cell-survival assays
gp130 LIFR LIFR/gp130 hOSMR/gp130
Wild-type – – 1.0 1.0 1.0 1.0 N terminus of helix D F160A 3 65 1.1 >100 >10,000 >10,000 K163A 3 42 0.9 >100 >100 >10,000 F160A/K163A 3 – 1.0 >100 >10,000 >10,000 N-terminal loop S7A 2 100 1.1 0.9 0.8 0.5 K8A 2 8 0.8 1.1 0.8 0.5 E9A 2 96 1.2 0.8 1.4 1.2 N terminus of helix A Y10A 2 35 3.4 7.2 2.1 0.9 R11A 2 74 1.0 1.9 0.8 0.5 V12A 2 55 1.0 1.2 0.7 0.7 Q16A 2 56 75.0 Q18 2 36 4.0 5.0 1.2 1.1 K19 2 79 1.0 2.0 1.4 2.0 Q20A 2 27 90.0 1.0 20.0 30.0 D22A 2 40 1.0 1.0 0.8 1.0 L23A 2 61 3.0 6.7 2.5 5.0 D26A 2 63 4.0 5.0 3.0 4.1 Helix C M113A 2 66 0.7 1.0 1.0 1.0 P116A 2 52 0.7 2.3 1.0 0.9 N117A 2 29 2.0 1.0 2.6 8.0 L119A 2 46 1.7 2.2 1.2 1.7 G120A 2 76 >100 1.8 60.0 60.0 G120Y 2 76 >100 1.8 >10,000 >10,000 N123A 2 61 8.0 1.0 3.3 10.0 N124A 2 31 >100 1.0 >10,000 >10,000
*RSA, the relative solvent accessibility of the sidechain. Mutants were tested for biological activity on Ba/F3 cells transfected with tested for inhibition of binding to immobilized gp130 and hLIFR. gp130/LIFR or hOSMR/gp130. Activity is expressed as the ratio EC50 Activity is expressed as the ratio IC50 mutant/IC50 WT hOSM. IC50 mutant/EC50 WT hOSM. EC50 refers to the concentration of mutant or refers to the concentration of mutant or WT protein required to elicit WT hOSM required to elicit a response equivalent to 50% of the 50% inhibition of binding of WT biotinylated hOSM. Mutants were also maximal response to hOSM. human OSMR/human gp130. The results for each mutant WT hOSM in the ability to interact with LIFR (data not are summarised in Table 3 and data from selected mutants shown), but greatly reduced activity in the ability to bind are shown in Figure 3. gp130 or stimulate the multiplication of gp130/LIFR (Figure 3b) or gp130/OSMR (Figure 3c) transfected BAF Identification of site 2 cells. A comparable rank order of activity was observed in The interaction of hLIF with gp130 (via site 2) has been both gp130-based solid-phase assays and bioassays; the previously shown to depend on a cluster of adjacent mutant Asn124Ala exhibited the greatest reduction in amino acids in the A and C helices, although the relative activity followed by Gly120Ala and Gln20Ala. We also significance of each amino acid could not be determined constructed a mutant in which Gly120 was replaced by [24]. This issue can be addressed by analysis of hOSM tyrosine (Gly120Tyr) thereby emulating the identity of which binds to gp130 with higher affinity than hLIF. the equivalent residue in CNTF that binds gp130 with Alanine mutations in the individual amino acids compris- very low affinity. This mutant also exhibited reduced ing the topologically equivalent region of hOSM ability to bind gp130 and stimulate the multiplication of revealed that the interaction between hOSM and gp130 transfected BAF cells and was significantly less active was critically dependent upon the identity of four struc- than the mutant Gly120Ala in both assay formats turally adjacent solvent-exposed residues, Gln20, Gly120 (Figures 3a–c). Collectively, these findings reveal that and Asn124 (Figure 3a), and, to a lesser degree, Gln16 the interaction of hOSM with gp130, the site 2 (Table 3). Each mutant exhibited equivalent activity to epitope, is governed by four residues located in a cluster Research Article Crystal structure of oncostatin M Deller et al. 869
Figure 3
(a) (d) (b) (e)