PMC:3405059 / 23396-30114 JSONTXT

{"project":"2_test","denotations":[{"id":"22848655-15167892-88310309","span":{"begin":2396,"end":2398},"obj":"15167892"},{"id":"22848655-17662939-88310310","span":{"begin":2402,"end":2404},"obj":"17662939"},{"id":"22848655-15167892-88310311","span":{"begin":2954,"end":2956},"obj":"15167892"},{"id":"22848655-15167892-88310312","span":{"begin":3214,"end":3216},"obj":"15167892"},{"id":"22848655-17662939-88310313","span":{"begin":3220,"end":3222},"obj":"17662939"},{"id":"22848655-15167892-88310314","span":{"begin":4589,"end":4591},"obj":"15167892"},{"id":"22848655-17662939-88310315","span":{"begin":4595,"end":4597},"obj":"17662939"},{"id":"22848655-17662939-88310316","span":{"begin":5194,"end":5196},"obj":"17662939"},{"id":"22848655-21114864-88310317","span":{"begin":6446,"end":6448},"obj":"21114864"},{"id":"T87479","span":{"begin":2396,"end":2398},"obj":"15167892"},{"id":"T9147","span":{"begin":2402,"end":2404},"obj":"17662939"},{"id":"T33959","span":{"begin":2954,"end":2956},"obj":"15167892"},{"id":"T86949","span":{"begin":3214,"end":3216},"obj":"15167892"},{"id":"T94736","span":{"begin":3220,"end":3222},"obj":"17662939"},{"id":"T4787","span":{"begin":4589,"end":4591},"obj":"15167892"},{"id":"T94360","span":{"begin":4595,"end":4597},"obj":"17662939"},{"id":"T33258","span":{"begin":5194,"end":5196},"obj":"17662939"},{"id":"T1202","span":{"begin":6446,"end":6448},"obj":"21114864"}],"text":"Comparison with Met Sema-PSI Structure\nThe RON and Met extracellular domains share ∼35% sequence identity. A structure-based sequence alignment of RON and Met Sema-PSI shows that, by and large, the secondary structural elements are conserved (Figure 2). The loops connecting the secondary structure elements are less conserved and contain multiple insertions and deletions. RON Sema loops contains α-helices that are absent in Met (α1D, α2B, α3B and αEx2; Figure 2), while the Met Sema loops have two β-strands that are absent in RON (β1D’ and β3D; Figure 2). The superposed structures show that the core β-sheets of the RON and Met β-propellers are well aligned but many of the surface loops adopt different conformations (Figure 3).\n10.1371/journal.pone.0041912.g003 Figure 3 Comparison of RON and Met structures.\n(A) Stereoscopic representation of superposed RON Sema-PSI (blue) and Met Sema-PSI (PDB entry codes 2UZX (gold) and 1SHY (pink) structures, viewed down the β-propeller as in Figure 1A. The Sema domains were superposed. (B) The superposed RON and Met, highlighting the loop connecting β-strands 1D and 2A and (C) highlighting the extrusion regions. Disulfide bonds of RON Sema are colored gold and those of Met Sema (PDB code 1SHY), red. The gold and red arrows highlight the locations of alternative disulfide linkages in RON and Met, respectively. RON and Met Sema domains contain 15 cysteine residues that form disulfide linkages (Figure 1A, 2). Three disulfide bonds are conserved (Cys135–Cys143, Cys300–Cys367, and Cys174–Cys177 in RON and Cys133–Cys141, Cys298–Cys363 and Cys172–Cys175 in Met). They link the intra β-strands 2B and 2C, the inter blade β-strands 4D and 5C, and a 20 residue loop connecting blades 2 and 3. However, four other conserved cysteine residues (Cys101, Cys104, Cys107 and Cys162 in RON Sema and Cys95, Cys98, Cys101 and Cys160 in Met Sema) form two different pairs of disulfide bonds (Figure 2, 3B). In RON, the linkages are between Cys101 and Cys104 located on the α-helical turn containing loop that connects β-strands 1D and 2A, and between Cys107 on the same loop and Cys162 located on β-strand 2D (Figure 3B). Alternative disulfide pairings have been reported for the analogous Met Sema loop in the Met/HGFβ structure (Cys95–Cys101 and Cys98–Cys160), but not in the Met/InlB structure where this loop was disordered and Cys160 was unpaired [63], [71]. The alternative disulfide bond and shorter 1D–2A loop of the RON Sema domain lead to a compact loop, whereas a longer loop in Met Sema lacks α-helical turn and folds into a flexible loop that extended above the core of β-propeller (Figure 3B) (Stamos et al., 2004). One more conserved cysteine in RON and Met Sema is located near the respective N-termini (Cys29 in RON and Cys26 in Met) (Figure 2). RON Cys29 and Met Cys26 are predicted to form an interdomain disulfide bond with the conserved cysteine in IPT1 domain (RON Cys590 and Met Cys584) [63]. In the RON Sema-PSI structure, Cys29 has been removed by proteolysis and Cys590 is located within the disordered RON IPT1 fragment. The putative Met Cys26–Cys584 inter-domain disulfide bond was also not observed in either Met/HGFβ or Met/lnlB structures [63], [71].\n10.1371/journal.pone.0041912.g004 Figure 4 Crystal packing generates a RON homodimer interface that overlaps with the putative MSPβ binding site predicted based on the Met/HGF structure.\n(A) Left panel: Surface and ribbon representations of symmetry-related RON Sema-PSI molecules. Right panel: Close-up view of the interface and the molecules rotated by ∼90°. (B) Surface and ribbon representation of the modeled RON Sema-PSI/MSPβ complex derived based on the free MSPβ (PDB code 2ASU) and RON Sema-PSI structures superposed onto the structure of Met Sema-PSI/HGFβ (PDB entry 1SHY). The molecular surfaces of RON Sema-PSI (blue) and MSPβ (pink) are shown in transparent colors and secondary structural elements are shown in ribbon representation. (C) Stereoscopic representations of the RON Sema homodimer interface residues generated by crystal packing. The two subunits are colored gray and sky blue. Selected amino acids are colored in the atomic color scheme: red, oxygen; blue, nitrogen; dark yellow, sulfur; bright yellow, acetate carbon. Two more RON Sema disulfide bonds are located on the large extrusion region (Cys385–Cys407 and Cys386–Cys422). These cysteine residues are not conserved in Met Sema. Instead, the extrusion of Met Sema contains a single disulfide bond (Cys385–Cys397) and an unpaired Cys409 in the disordered loop (Figure 2 and 3C) [63], [71]. Another non-conserved Cys282 in Met Sema is positioned at the end of β-strand 4C near the extrusion region. In all, the alternate disulfide bonding patterns in the 1D–2A loop and the extrusion regions of RON and Met Sema domains define specificity determinants, which allow RON and Met receptors to interact exclusively with either MSP or HGF, respectively.\nAs reported earlier, when the two available structures of Met are compared, the superposed structures of the Met/HGFβ and Met/InlB complexes reveal different orientations assumed by the Met PSI with respect to the aligned Sema domains [71]. The C-termini of the Met PSI in these structures are displaced by ∼15 Å and are rotated by ∼60° with respect to a common axis defined by the region linking the Sema and PSI domains. The RON PSI module adopts yet another orientation (Figure 3A). The RON PSI is flanked on one side by the Met PSI from the Met/HGFα complex with ∼8 Å displacement, and on the other side by the Met PSI from the Met/InlB complex with ∼10 Å displacement (Figure 3A). Similarly to Met, the conserved Gly524 and Gly526 located at the Sema-PSI linker region modulate the relative orientation of RON PSI (Figure 2). Moreover, both structures of Met complexes and RON Sema-PSI structure lack the disulfide bond predicted to link the disordered/degraded N-terminal region with the IPT1 domain. The relative orientations of the Sema, PSI, and IPT1 domains might still be different in the presence of this interdomain disulfide linkage.\nThe ability of RON and Met ectodomains to adopt multiple interdomain orientations may play critical roles in selective ligand binding and receptor dimerization. For instance, the RONΔ160 splice variant, lacking the 103 residue long IPT1 domain, readily forms dimers in the absence of MSP and displays constitutive phosphorylation activity [49]. In the absence of IPT1 domain, the adoptable PSI hinge may provide a mechanism for reorientation of the remaining RON ectodomains that allow MSP-independent receptor dimerization and concomitant juxtaposing of the intracellular kinase domains for autophosphorylation."}