Crystal Structure and Functional Dissection of the Cytostatic Cytokine

View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector 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.

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