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EMBO J. Jan 11, 2006; 25(1): 80–87.
Published online Dec 15, 2005. doi:  10.1038/sj.emboj.7600912
PMCID: PMC1356355

Structural basis for Gas6–Axl signalling


Receptor tyrosine kinases of the Axl family are activated by the vitamin K-dependent protein Gas6. Axl signalling plays important roles in cancer, spermatogenesis, immunity, and platelet function. The crystal structure at 3.3 Å resolution of a minimal human Gas6/Axl complex reveals an assembly of 2:2 stoichiometry, in which the two immunoglobulin-like domains of the Axl ectodomain are crosslinked by the first laminin G-like domain of Gas6, with no direct Axl/Axl or Gas6/Gas6 contacts. There are two distinct Gas6/Axl contacts of very different size, both featuring interactions between edge β-strands. Structure-based mutagenesis, protein binding assays and receptor activation experiments demonstrate that both the major and minor Gas6 binding sites are required for productive transmembrane signalling. Gas6-mediated Axl dimerisation is likely to occur in two steps, with a high-affinity 1:1 Gas6/Axl complex forming first. Only the minor Gas6 binding site is highly conserved in the other Axl family receptors, Sky/Tyro3 and Mer. Specificity at the major contact is suggested to result from the segregation of charged and apolar residues to opposite faces of the newly formed β-sheet.

Keywords: immunoglobulin-like domain, laminin G-like domain, receptor tyrosine kinase, site-directed mutagenesis, X-ray crystallography


Receptor tyrosine kinases (RTKs) play critical roles in the control of many cellular processes, such as the cell cycle, cell metabolism and survival, proliferation and differentiation, and cell migration (reviewed in Schlessinger, 2000; Gschwind et al, 2004). The prototypical RTKs are activated by growth factors, which promote receptor dimerisation and, in turn, autophosphorylation of tyrosine residues within the cytosolic domain. Binding of signalling proteins to these phosphorylated tyrosine residues then leads to downstream signalling. Axl and its homologues, Sky/Tyro3 and Mer, constitute an RTK subfamily characterised by an extracellular domain resembling cell adhesion molecules, consisting of two immunoglobulin-like (IG) domains followed by two fibronectin type 3-like domains (Janssen et al, 1991; O'Bryan et al, 1991; Rescigno et al, 1991). Axl family RTKs are unique in that they are activated by Gas6, a member of the vitamin K-dependent protein family that resembles blood coagulation factors rather than typical growth factors (Ohashi et al, 1995; Stitt et al, 1995; Varnum et al, 1995; Mark et al, 1996). Gas6 consists of a γ-carboxyglutamate (Gla)-rich domain that mediates binding to phospholipid membranes, four epidermal growth factor-like domains, and two laminin G-like (LG) domains (Manfioletti et al, 1993). Gas6 is ~40% identical to protein S, a negative regulator of blood coagulation that can also activate Axl RTKs under certain nonphysiological conditions (Godowski et al, 1995).

The biological consequences of Axl activation are complex. Axl was initially identified as a transforming gene product (Janssen et al, 1991; O'Bryan et al, 1991), and Axl expression is indeed upregulated in human tumours (Nakano et al, 2003; Sun et al, 2004). Many studies have shown that Gas6–Axl signalling promotes cell survival and growth in vitro (e.g. Nakano et al, 1995; Goruppi et al, 1996; Li et al, 1996; O'Donnell et al, 1999; Shankar et al, 2003). Recent gene knockout studies have revealed fundamental roles of Axl RTKs in spermatogenesis (Lu et al, 1999), immunity (Lu and Lemke, 2001; Scott et al, 2001), platelet function (Angelillo-Scherrer et al, 2001, 2005) and kidney pathology (Yanagita et al, 2002). The role of Gas6–Axl signalling in platelets is particularly interesting. Cross-talk between Axl RTKs and αIIbβ3 integrin, via PI3 kinase and Akt, contributes to platelet activation and thrombus stabilisation. Importantly, blocking of Gas6–Axl signalling in mice prevents thrombosis without causing the bleeding side effects that blight other antithrombotic therapies (Angelillo-Scherrer et al, 2005; Gould et al, 2005). A better understanding of the molecular recognition events leading to Axl activation is thus of considerable interest.

Previous studies have shown that the Axl binding site of Gas6 is fully contained within the C-terminal LG domain pair (Gas6-LG) (Mark et al, 1996; Tanabe et al, 1997). A structure determination of Gas6-LG revealed a V-shaped arrangement of two canonical LG domains, reinforced by an interdomain calcium binding site (Sasaki et al, 2002). Like in the related LG pair of the laminin α2 chain (Tisi et al, 2000), the N-terminal segment of Gas6-LG is disulfide-bonded to the second LG domain, LG2. A hydrophobic patch on LG2 was tentatively assigned as an Axl binding region, but how Gas6 binds to and activates Axl remained unclear (Sasaki et al, 2002). A recent structure determination of the IG domains of Sky also failed to give insight into this critical question (Heiring et al, 2004). Here we report the crystal structure and mutational analysis of a minimal Gas6/Axl complex. Our results demonstrate that Gas6 initiates signalling by crosslinking its receptor by an unusual mechanism of β-sheet juxtaposition. The Gas6/Axl structure provides a framework for future functional studies and should be useful in developing reagents blocking aberrant Axl signalling in human diseases.


Overall structure of the minimal Gas6–Axl complex

The soluble Axl ectodomain binds Gas6-LG with nanomolar affinity (Sasaki et al, 2002) and 1:1 stoichiometry, as determined by analytical ultracentrifugation (data not shown). We found that an Axl fragment spanning the two N-terminal IG domains and lacking carbohydrate modifications (Axl-IG) retains full Gas6-LG binding activity. We crystallised the human Gas6-LG/Axl-IG complex and have determined its structure at 3.3 Å resolution (Rfree=0.265). The asymmetric unit of the crystals contains a symmetric 2:2 Gas6-LG/Axl-IG assembly, in which the Axl-IG molecules are located on the outside and are linked by two Gas6-LG molecules (Figure 1). There are no direct ligand/ligand or receptor/receptor contacts. Only LG1 of Gas6 is involved in Axl binding, whereas both IG1 and IG2 of Axl are involved in Gas6 binding. The three predicted glycosylation sites of Axl-IG (Asn36Axl, Asn150Axl and Asn191Axl) are distant from the Gas6-LG molecules in the 2:2 complex, consistent with our finding that Axl glycosylation has no influence on high-affinity Gas6 binding.

Figure 1
Overall architecture of the Gas6-LG/Axl-IG complex. Shown are three orthogonal views. (A) Top view, towards the cell membrane harbouring the receptor. (B) Side view, with the cell membrane at the bottom. (C) Front view, in the direction indicated by the ...

Structure of Gas6-LG and Axl-IG in the complex

The structure of Gas6-LG in the complex is very similar to that of free Gas6-LG (Sasaki et al, 2002), but there is a small reorientation by 9° in the interdomain angle, and two previously disordered segments in LG1Gas6 become ordered upon complex formation (see below). The two IG domains of Axl-IG are arranged in a fully extended fashion that differs from the bent conformation seen in the IG pair of the homologous receptor, Sky (Heiring et al, 2004) (Figure 2). IG1Axl belongs to the intermediate (I) set of the IG superfamily, with the two β-sheets showing the characteristic ABED and A′GFCC′ topology (Chothia and Jones, 1997). The putative glycosylation site at Asn36Axl is located in strand A′. IG2Axl also resembles the I set, but strand D is missing, similar to the situation in ICAM-1 (Casasnovas et al, 1998). Putative glycosylation sites are located in strand B (Asn150Axl) and the E-F loop (Asn191Axl). A comparison of the Axl and Sky structures reveals remarkable differences in their IG1 domains (r.m.s. deviation 2.4 Å for 79 Cα atoms; Figure 2B), whereas their IG2 domains are quite similar (r.m.s. deviation 0.74 Å for 78 Cα atoms). IG1 of Sky deviates from IG1Axl and all other I-set members, in that strand G (rather than strand A) switches between the two β-sheets of the domain, hydrogen bonding first to strand B and then to strand F (Heiring et al, 2004). Another notable difference is that the IG pair of Sky is dimeric, with the ABED sheet of the IG1 domain forming the dimer interface (discussed below).

Figure 2
Comparison of Axl family members. (A) Comparison of the structures of Axl-IG (this work) and a Sky-IG monomer (Heiring et al, 2004). The two structures were superimposed using their IG1 domains. Axl is shown in yellow (IG1) and brown (IG2), Sky in light ...

Two types of Gas6/Axl contacts

There are two distinct ligand/receptor contacts in the 2:2 Gas6-LG/Axl-IG complex described here. The major contact, between LG1Gas6 and IG1Axl, buries 2366 Å2 of solvent-accessible surface area and, given its size, is tentatively assigned as the high-affinity interaction site (confirmed below). The minor contact, between LG1Gas6 and IG2Axl, buries 765 Å2. Remarkably, both types of contact feature the juxtaposition of edge β-strands, so that continuous β-sheets are formed across both Gas6/Axl contacts (Figure 1C). Neither contact displays particularly good shape complementarity: when evaluated with the program SC (CCP4-Collaborative Computing Project No. 4, 1994), the major contact scores 0.54, and the minor contact 0.63 (a score of 1 indicating perfect complementarity of the interacting surfaces). In the crystal lattice, Gas6-LG/Axl-IG complexes are arranged in infinite linear arrays, with a two-fold noncrystallographic symmetry axis producing an Axl/Axl contact that buries 2 × 814 Å2 of solvent-accessible surface (SC score 0.48). The resulting ‘Axl dimer' does not resemble the much tighter Sky dimer (Heiring et al, 2004) and is probably nonphysiological, given the paucity of specific interactions and the presence of Ni2+ ions in the interface (Supplementary Figure S1).

Major Gas6/Axl contact

The core of the major Gas6/Axl contact is formed by the antiparallel interaction of strand B of LG1Gas6 and strand D of IG1Axl (six main-chain hydrogen bonds). Formation of a continuous β-sheet across the major Gas6/Axl contact leads to a striking segregation of charged and apolar residues. On one face of the β-sheet, an extensive network of charged residues is formed, with Arg308Gas6, Arg310Gas6 and Lys312Gas6 interacting with Glu56Axl and Glu59Axl; the latter two residues are contributed by the B-C loop located above strand D of IG1Axl (Figure 3A). The two N-acetylglucosamine moieties attached to Asn420Gas6 pack against Arg310Gas6 and are close to Axl, but appear to make no specific interactions with the receptor. The opposite face of the major contact is devoid of charged residues and is dominated by apolar interactions, most prominently between Thr75Axl, Thr77Axl, Val79Axl, Ile90Axl and Val92Axl on the one hand, and Ile307Gas6, Leu309Gas6, Phe311Gas6, Thr457Gas6, Ile458Gas6, Thr461Gas6 and Met468Gas6 on the other hand (Figure 3B). Gas6 residues 457–464 are part of a short α-helix that was not seen in free Gas6-LG (Sasaki et al, 2002). This helix therefore appears to become ordered only upon receptor binding, although its association with Axl is remarkably loose (there are only two van der Waals contacts closer than 4 Å). Finally, the A–B loop in LG1Gas6 is also disordered in free Gas6-LG, but contributes two Axl-binding residues, Arg299Gas6 and Ser302Gas6, in the complex.

Figure 3
Detailed structure of the Gas6/Axl contact sites. (A) Front view of the major contact, in a direction similar to Figure 1C, showing the polar β-sheet surface. (B) Back view of the major contact, showing the apolar β-sheet surface. (C) ...

We previously reported that mutation of Leu620Gas6 and Tyr660Gas6 in LG2Gas6 has a modest effect on Axl binding and activation (Sasaki et al, 2002), yet we now see that LG2Gas6 is not directly involved in receptor binding. However, Leu620Gas6 and Tyr660Gas6 are close to the Axl-binding A-B loop in LG1Gas6 (12 and 3.5 Å, respectively), suggesting that their mutation may have affected the Gas6/Axl interaction at the major site in an indirect manner.

Minor Gas6/Axl contact

The minor Gas6/Axl contact is formed between strands J of LG1Gas6 and strand G of IG2Axl, with additional contributions from the domain linker of Axl-IG (Figure 3C). Apart from three main-chain hydrogen bonds, there is only one polar interaction in this region, between Asp398Gas6 and Lys204Axl. Additionally, there are several van der Waals contacts that may impart specificity: most notably, Leu131Axl is inserted into a pocket delineated by Ala399Gas6 and Lys402Gas6, and the Cβ atom of Ala404Gas6 makes a close contact with Cα of Arg210Axl.

Both Gas6/Axl contacts are required for receptor activation

As it is often difficult to distinguish crystal lattice contacts from genuine, physiological ligand/receptor interactions, we sought to confirm our interpretation of the Gas6-LG/Axl-IG structure by designing mutations that would disrupt specifically the major or minor contact in the 2:2 Gas6-LG/Axl-IG complex, or were predicted to have no effect (control mutations). The choice of mutations was complicated by the fact that both Gas6/Axl contacts prominently involve β-strand interactions, which are made by the polypeptide backbone and therefore not easily disrupted by mutagenesis.

At the major contact, mutation of Glu59Axl and Thr77Axl dramatically reduced Gas6 binding both in a solid-phase assay and when measured by surface plasmon resonance (Table I). Likewise, mutation of Arg310Gas6 and Lys312Gas6 all but abolished Axl binding in both assays. Thus, the major contact in the Gas6-LG/Axl-IG complex indeed corresponds to the high-affinity interaction site. We were surprised that Glu56Axl in the B-C loop of IG1Axl does not seem to be required for a high-affinity interaction, given that this residue makes a specific electrostatic interaction with Gas6. However, Glu56Axl remains partly exposed in the complex, and its role may be redundant (see below).

Table 1
Mutational analysis of the Gas6/Axl interaction

To test whether the major contact is required for Gas6–Axl signalling, we compared wild-type and mutant Gas6-LG proteins in a receptor activation assay, using a tumour cell line expressing Axl (Sasaki et al, 2002). Mutation of Arg310Gas6 and Lys312Gas6 reduced Axl phosphorylation to background levels (Figure 4), showing that the major Gas6/Axl contact is required for productive signalling. Unfortunately, the effect of Axl mutations on signalling could not be studied, since overexpression of Axl in a number of cell lines resulted in constitutive receptor activation in the absence of Gas6 (data not shown).

Figure 4
Axl activation by wild-type and mutant Gas6-LG. Human U-118 glioblastoma cells were incubated with 200 ng/ml Gas6-LG proteins for 15 min, the cells lysed, and the Axl receptor immunoprecipitated. Blots were probed with an anti-phosphotyrosine antibody, ...

Mutation of Axl or Gas6 at the minor contact did not appreciably affected their high-affinity interaction, as expected (Table I). Crucially, however, mutation of Ala404Gas6 resulted in a marked reduction of Axl activation (Figure 4). Ala404Gas6 is fully exposed in free Gas6, but becomes partly buried at the minor contact in the complex. Thus, the reduced activity of the Gas6-LG A404V mutant demonstrates that the minor contact is functionally important and not a crystal lattice artefact. In support of this conclusion, a monoclonal antibody that partially blocks the Gas6/Axl interaction recognises Gas6 residues 393–414, which contain the Axl-binding β-strand J and Ala404Gas6 (Fisher et al, 2005). The combined data demonstrate that the 2:2 Gas6/Axl complex seen in our crystals represents a physiologically relevant assembly.

Sequence conservation of Gas6/receptor contacts

All Gas6 regions involved in Axl binding, including the glycosylation site at Asn420Gas6 in the major contact, are strictly conserved in vertebrate Gas6 sequences (not shown). Comparing Axl with its family members Sky and Mer, we were surprised to find that only the minor Gas6 binding site in IG2Axl is highly conserved, whereas the major site in IG1Axl is not well conserved in Sky and Mer (Figure 2C). However, even though individual amino acids are not conserved, pertinent characteristics of the major Gas6 binding site in Axl appear to be conserved, at least in Sky (for which structural information is available; Heiring et al, 2004): the B-C loop of IG1 contains negatively charged residues, and strand D is quite long and features an unusually apolar surface that is contiguous with exposed apolar residues on strand E. The hydrophobic nature of the D-E surface is illustrated by the fact that, in the absence of Gas6, the Sky IG pair forms dimers in which the Gas6-binding D-E surface in IG1 is occluded in the dimer interface. This dimer may be an artefact of bacterial expression, since a putative glycosylation site is buried in the dimer interface (Figure 2C). Importantly, it has been shown that the dimer dissociates upon addition of Gas6-LG, resulting in the formation of a ligand/receptor complex with 1:1 stoichiometry (Heiring et al, 2004). Modelling shows that monomeric Sky is readily accommodated at the major Axl binding site of Gas6-LG (not shown).

With respect to the third Gas6 receptor, Mer, it is difficult to discern any conservation of Gas6 binding features at the major contact, and this may be correlated with the reduced affinity of Mer for Gas6 (Nagata et al, 1996). We cannot exclude that Mer (or, indeed, Sky) binds Gas6 in a different manner than Axl, but we think this is unlikely. It is also worth noting that all putative N-linked glycosylation sites in Sky and Mer (a total of seven sites, only one of which is shared with Axl) map to solvent-exposed positions in the Gas6-LG/Axl-IG structure. Given that all three Axl family receptors have multiple glycosylation sites within their IG domains (Figure 2C), the complete absence of such sites from the predicted Gas6 binding regions supports a conserved ligand-binding mode.


RTK activation is believed to result from ligand-induced receptor dimerisation, leading to the autophosphorylation of multiple tyrosine residues in the cytosolic domains, and to downstream signalling (Schlessinger, 2000). Indeed, the close approximation of (at least) two receptors is a feature of all known structures of ligand/RTK complexes, but the mechanism by which receptor dimerisation is brought about is different in each case (Schlessinger, 2000; Wiesmann et al, 2000; Himanen et al, 2001; Burgess et al, 2003). Additional complexity is introduced by a variety of autoinhibitory mechanisms that help maintain RTKs in an inactive state in the absence of ligand, and the existence of (obligate) coreceptors. The architecture of the heterotetrameric Gas6/Axl complex described here most closely resembles that of the ephrin/Eph complex (Himanen et al, 2001), even though the two systems are completely different in their domain organisation. Both complexes are circular 2:2 assemblies with two distinct ligand/receptor interfaces of different affinity, and no direct ligand/ligand or receptor/receptor contacts. Like Eph signalling, Axl signalling is likely to be initiated by the formation of a high-affinity 1:1 ligand/receptor complex. Gas6/Axl complex formation may be driven initially by the combination of charge complementarity and hydrophobicity at the major contact site, followed by the receptor-induced freezing of mobile Gas6 regions. Lateral diffusion of 1:1 complexes would then lead to the formation of a minimal 2:2 Gas6/Axl signalling complex. Further aggregation of 2:2 complexes has been demonstrated to be required for Eph signalling (Smith et al, 2004). Whether Axl/Axl interactions contribute to signalling remains to be established.

The central role of hydrogen pairing between β-strands in both types of Gas6/Axl contacts is surprising, given that these interactions are not sequence-specific. At the major Gas6/Axl contact, the striking segregation of polar/ionic and apolar side chain interactions to opposite surfaces of the newly formed β-sheet appears to ensure the specific recognition of the cognate ligand. The precise nature of many of these interactions may be less critical, as suggested by the modest shape complementarity at this contact and the lack of sequence conservation of many Gas6 binding residues in the other Axl family receptors, Sky and Mer. The minor Gas6/Axl contact is likely to be very weak and may owe its existence to the high cooperativity of forming a circular 2:2 complex. Nevertheless, the minor contact is critical for signal transduction, as suggested by its striking sequence conservation and experimentally verified by mutagenesis of Ala404Gas6. Molecules that are able to inhibit Gas6/Axl signalling are potentially useful in controlling thrombosis (Angelillo-Scherrer et al, 2005; Gould et al, 2005), and our structure provides a framework for the rational design of such blocking agents. Targetting the Gas6/Axl contacts seen in our structure by small molecule antagonists may prove challenging, however: one contact is very large, and the other involves little more than the interaction of two solvent-exposed β-strands.

Several questions remain unanswered by our structure of a minimal Gas6/Axl complex. The Gla domain of Gas6 is dispensable for Axl activation in cultured cells, but its role in vivo has not been studied. The Gla domain may help recruit Gas6 to the cell surface harbouring its receptor. Model building shows that this is sterically feasible; the EGF-like domain preceding LG1Gas6 could interact with the hydrophobic patch in LG2Gas6, previously speculated to be involved in receptor binding (Sasaki et al, 2002). Alternatively, Gas6 could be bound to a different cell membrane than its receptor, similar to the ephrin/Eph situation. The physiological role, if any, of receptor dimerisation also remains to be studied. The fully glycosylated Axl ectodomain forms dimers with a dissociation constant of ~10 μM (our unpublished results), but whether this association occludes the major Gas6 binding site, as in the Sky structure (Heiring et al, 2004), is not known. To understand Gas6/Axl signalling in a cellular context, it will be important to determine the oligomeric state of Axl within the cell membrane and how it is affected by Gas6 binding. Our structure and mutational validation of a minimal Gas6/Axl complex has revealed the unique mechanism by which Axl family RTKs are dimerised by Gas6, and provides a solid basis to guide future investigations into this system.

Materials and methods

Protein production and mutagenesis

Human Gas6-LG was expressed in human embryonic kidney (293-EBNA) cells and purified as described (Sasaki et al, 2002). Point mutations were introduced by fusion PCR and confirmed by sequencing. All mutants behaved indistinguishable from wild-type Gas6-LG during purification. Soluble Axl-IG was expressed in Escherichia coli BL21 cells, by secretion into the periplasmic space. DNA coding for Axl residues 26–220 (sequence numbering includes the signal peptide as defined in SwissProt entry P30530) was obtained by PCR from a full-length human Axl cDNA and cloned into pASK-IBA12 (N-terminal Strept-tag II) or pASK-IBA32 (C-terminal His-tag) (IBA GmbH, Göttingen, Germany). Point mutations were introduced using a site-directed mutagenesis kit (Stratagene, CA, USA) and confirmed by sequencing. Proteins were purified according to the manufacturer's instructions. The Strept-tag was removed by thrombin digestion. The untagged Axl-IG was purified by gel filtration (Superose 12 HR 16/50) and mixed with Gas6-LG in a 1:1 molar ratio. The resulting Gas6-LG/Axl-IG complex was purified by gel filtration prior to crystallisation.

Binding and activation assays

The solid-phase assay and surface plasmon resonance experiments were carried out as described previously (Sasaki et al, 2002). Soluble Axl ectodomain (Sasaki et al, 2002) or Axl IG1–2 and its mutants (His-tagged proteins) were immobilised, and Gas6-LG and its mutants were used as soluble ligands. Axl activation by wild-type and mutant Gas6-LG in human U-118 glioblastoma cells was also carried out as described previously (Sasaki et al, 2002).

Crystal structure determination

For crystallisation, the Gas6-LG/Axl-IG complex was concentrated to 10 mg/ml in 0.025 M Na-HEPES pH 7.5, 0.15 M NaCl, 1 mM calcium acetate. Needle-shaped crystals were obtained at room temperature by the hanging drop method, using 0.1 M Tris–HCl pH 8.5, 0.6–0.8 M Li2SO4, 2 mM NiSO4 as precipitant. The crystals were flash-frozen in mother liquour supplemented with 30% glycerol. X-ray diffraction data were collected at 100 K on beamline ID23-1 at the ESRF (Grenoble, France) using a Marmosaic 225 CCD detector (λ=0.9794 Å). The crystals diffracted extremely weakly and data were collected using an unattenuated beam and 20 s exposures per 0.5° frame. There are two copies of a 1:1 Gas6-LG/Axl-IG complex in the asymmetric unit of the hexagonal crystals, related by a noncrystallographic two-fold symmetry axis perpendicular to the crystallographic six-fold axis. The solvent content of the crystals is unusually high at 78%, explaining the weak diffraction. The diffraction data were processed with MOSFLM (http://www.mrc-lmb.cam.ac.uk/harry/mosflm/) and programs of the CCP4 suite (CCP4-Collaborative Computing Project No. 4, 1994). The structure was solved by molecular replacement with PHASER (Storoni et al, 2004), using the Gas6-LG (Sasaki et al, 2002) and Sky-IG (Heiring et al, 2004) structures as search models; the IG domains of Sky had to be placed individually to obtain a solution. The structure was rebuilt with O (Jones et al, 1991) and refined with CNS (Brunger et al, 1998), using tight noncrystallographic symmetry restraints. The final model includes: Gas6 residues 279–493, 504–541, and 550–676, with two N-acetylglucosamine moieties attached to Asn420Gas6; Axl residues 27–217; two calcium ions, two nickel ions, and one sulfate ion. Data collection and refinement statistics are summarised in Table II. Solvent-accessible areas were calculated with the CCP4 program AREAIMOL. The figures were made with PYMOL (http://www.pymol.org).

Table 2
Crystallographic statistics

Supplementary Material

Supplementary Figure S1


We thank Jürgen Engel and Ariel Lustig (Biozentrum Basel, Switzerland) for the analytical ultracentrifugation experiments; Xavier Thibault and the staff at ESRF (Grenoble, France) and Federico Carafoli for help with X-ray data collection; Peter Brick, Jason Howitt and Birgit Leitinger for critical reading of the manuscript. This work was supported by a Wellcome Senior Research Fellowship to EH (ref 54334) and a DFG grant to RT (ref Ti95/8-2).

Accession code The coordinates and structure factors of the Gas6-LG/Axl-IG complex have been deposited in the Protein Data Bank (accession code 2c5d).


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