Ligand-independent RON Dimerization
In addition to the MSP-mediated RON receptor activation, ligand-independent RON dimerization and constitutive phosphorylation activity have been observed in numerous cancer and tumor cells which over expressed full length RON receptor and expressed the RONΔ160 splice variant [20], [72], [73]. RON intermolecular interactions generated by the crystal packing reveal a potential mode of ligand-independent dimerization, mediated by the Sema domain (Figure 4A). This is the most extensive crystallographic related RON Sema-Sema interface with ∼960 Å2 embedded surface area, a rather large interface for typical crystal contacts [74]. Therefore, this crystal-generated interface may have a functional role at the cellular level. Multiple electrostatic interactions between the bottom surface loops of blades 3–4 and the edge residues of the extrusion region are involved, and these are repeated twice due to the crystal 2-fold symmetry axis. Two striking networks stand out within this dimer interface. First, Glu387 forms an intermolecular salt bridge with Arg220, and the carboxylate group of Glu387 also interacts with the NH of Ala223 of the neighboring molecule (Figure 4C). The guanidinium groups of Arg220 and Arg423 of the partner Sema interact with an interface sulfate ion (present in the crystallization solution). The positioning of the sulfate ion is further stabilized by the hydrogen bonding with the hydroxyl group of Ser421 and by the main chain NH groups of Cys422 and Arg423. The arrangement of this sulfate-binding site appears optimal for accommodating a phosphoryl group on Ser421, although the physiological phosphorylation state of this residue is unknown.
A second intermolecular electrostatic cluster at the crystallographic RON Sema-PSI dimer interface comprises three carboxylate groups; two from one subunit (Glu289 and Asp292) and the third from the second subunit (Glu287) (Figure 4C). This particular type of proton sharing interaction between carboxyl-carboxylate groups is favorable only at pH below 6 (Sawyer and James, 1982), consistent with the acidic condition (pH 4.6) used to obtain the RON Sema-PSI crystals. Multitude secondary and tertiary shells of interactions support the formation of both electrostatic clusters. Finally, an acetate ion (pH buffering component of the crystals) is located on a special crystallographic 2-fold symmetry position, bridging two His242 imidazole groups, albeit at somewhat remote distances (3.4 Å). The pH-dependent intermolecular interactions, described above, suggest that ligand-independent homodimerization of RON may play a functional role in the acidic extracellular microenvironments often associated with tumors and under other cellular acidosis conditions [75], [76].
This crystallographically observed RON Sema-PSI homodimer, generated by a Sema-Sema interface, might pertain to the mechanism of ligand-independent constitutive activity of RONΔ160 splice variant and its inhibition by RONΔ85 [16]. RONΔ160, lacking the 103-residue IPT1 domain, is a cell surface receptor that readily forms homodimers and is constitutively active in the absence of MSP. RONΔ85 splice variant, on the other hand, is a soluble protein comprising only the Sema, PSI, and 64 amino acid residues of IPT1 domain. The addition of RONΔ85 reduced the levels of phosphorylated RONΔ160 as well as those of phosphorylated downstream signaling molecules, ERK1/2 and Akt, in a dose-dependent manner [16]. The co-immunoprecipitation experiments revealed a direct association between RONΔ160 and RONΔ85 molecules, and RONΔ160 dimerization was lower in cells treated with RONΔ85 [16], [20]. MSP did not prevent the RONΔ85 inhibition; thus, the dominant negative effect appears to be a direct consequence of RONΔ85 binding to the membrane-bound RONΔ160 [16]. Ma and colleagues suggested the Sema-Sema interaction between RONΔ85 and RONΔ160 as the possible mechanism of inhibition, perhaps employing the Sema-Sema interface observed in the RON Sema-PSI structure (Figure 4A).
The full length RON also exhibits ligand-independent dimerization at high receptor density, which may be responsible for its constitutive activity in tumors [14], [40], [72], [73]. RONΔ90 splice variant, comprising Sema, PSI and 70 amino acids of IPT1, was shown to inhibit the MSP-induced RON phosphorylation activity and to attenuate the basal constitutive activity of RON in the absence of external MSP. RONΔ90, found in several glioblastoma cell lines, blocked both the MSP-induced migration and random motility of these cells [14]. Analogous to the interaction between RONΔ85 and RONΔ160 splice variants, we propose that RONΔ90 splice variant may sequester the full length RON as an inactive dimer using the mode of homodimerization seen in the crystals, thus exerting an antagonistic effect on cell migration.
In this crystal homodimer, the PSI motifs extend from their respective Sema domains in the same direction as expected for membrane-anchored receptors (Figure 4A). Approximately 50 Å separates the C-termini of the PSI domains, a reasonable distance that can be bridged by the IPT domains to bring together two membrane-spanning segments so that the intracellular kinase domains can interact and undergo constitutive autophosphorylation in trans.
RON Sema domain was identified as the high affinity binding site for MSPβ [33], [36]. We have mapped the high affinity MSPβ binding site on the RON Sema domain, based on the Met Sema-PSI/HGFβ structure and the structural homologies between the RON and Met receptors and their MSP and HGF ligands (Figure 4B). The model shows a region of the RON homodimer interface overlapping with a same region of RON Sema predicted to bind to MSPβ. The overlap between the binding regions lends support to our proposal that the crystallographically observed mode of RON Sema homodimerization represent the in vivo ligand-independent, constitutively activated RON homodimer. Similar modes of protein-protein interactions occur in the semaphorins and plexin receptors. That is, in semaphorins and plexins, the extrusion region of one Sema subunit interacts with a second homodimer subunit, or with the ligands or co-receptors [61], [62], [66], [67]. For example, using the same interface, plexin A2 dimer undergoes a partner switch to accommodate Sema6A dimer ligand, forming a 2∶2 signaling complex [62]. Although the arrangements of the RON, semaphorin, and plexin homodimers differ, all interfaces engage the extrusion region present in all Sema domains but structurally unique in each family member [62], [67], [69].
We note a second crystal packing interaction between symmetry-related RON Sema-PSI molecules involving a much smaller embedded surface area (∼390 Å2), mediated by hydrophobic interactions. Top surface loops connecting β-strands 4E–6A, 6B–6C and 6D–7A along with the N-glycans linked to Asn488 of RON Sema participate in formation of this Sema-Sema interface (data not shown). In this homodimer arrangement, the RON PSI motifs also extend from the respective Sema domains in the same direction and their C-termini are separated by ∼15–24 Å. The IPT domains of this dimer can make molecular contacts along the stalk of RON’s ectodomain. However, this interface only formed because the N-terminus of RON Sema had undergone proteolysis. Formation of such dimer would be blocked in the presence of the N-terminal residues.
In summary, the structure of RON Sema-PSI provides new insights into the features that define the MSPβ specificity and the possible mechanism of ligand-independent RON receptor activation. Analysis of RON mode of homodimerization and comparison with the semaphorins and plexin receptors suggests that all Sema-type proteins employ homodimerization interfaces that overlap with the ligand binding interfaces as a mechanism to regulate their signaling activities.