Little is known about the large ectodomain of MET, the product of the c-met protooncogene and receptor for hepatocyte growth factor͞scatter factor (HGF͞SF). Here, we establish by deletion mutagenesis that the HGF͞SF and heparin-binding sites of MET are contained within a large N-terminal domain spanning the ␣-chain (amino acids 25-307) and the first 212 amino acids of the ␤-chain (amino acids 308 -519). Neither the cystine-rich domain (amino acids 520 -561) nor the C-terminal half of MET (amino acids 562-932) bind HGF͞SF or heparin directly. The MET ectodomain, which behaves as a rod-shaped monomer with a large Stokes radius in solution, binds HGF͞SF in the absence or presence of heparin, and forms a stable HGF͞SF-heparin-MET complex with 1:1:1 stoichiometry. We also show that the ligand-binding domain adopts a ␤-propeller fold, which is similar to the N-terminal domain of ␣V integrin, and that the C-terminal half contains four Ig domains (amino acids 563-654, 657-738, 742-836, and 839 -924) of the unusual structural E set, which could be modeled on bacterial enzymes. Our studies provide 3D models and a functional map of the MET ectodomain. They have broad implications for structurefunction of the MET receptor and the related semaphorin and plexin proteins.Ig domain ͉ sema domain ͉ integrin ␣-chain ͉ hidden Markov models ͉ semaphorins R eceptor tyrosine kinases (RTKs) mediate intercellular signals, which are essential for the development and maintenance of the cells of multicellular organisms. The minimal domain structure of RTKs consists of an extracellular ligandbinding domain, a single transmembrane helix, and a cytoplasmic kinase domain. This minimal structure, however, is very rare, and, typically, the extracellular moiety of RTKs, the ectodomain, consists of complex and distinctive domain sets, which enable classification of the RTKs in different families (1).There is a strong preference for certain domains to occur in the ectodomain of RTKs. The fibronectin type-3 (FN-3) domain, for example, is present as two copies in the large Eph receptor family, three copies in the insulin and IGF-1 receptors, and at least seven copies in the rod outer segment receptor (1). Cystine-rich domains of variable length are also commonly found in RTKs.A large number of RTKs contain Ig domains and the ectodomain of certain families consists solely of Ig domains: the fibroblast growth factor (FGF) receptors contain two or three, depending on RNA splicing, the platelet-derived growth factor (PDGF), colony-stimulating factor 1 (CSF1), KIT, and FLT kinase͞serine-threonine kinase 1 (FLK2͞STK1) receptors contain five, and the FMS-like (FLT1), FLK1, FLT4, and cholecystokinin 4 (CCK4) receptors contain seven (1). Ig domains can also be present in combination with FN-3, cystine-rich, or other domains (1). Interestingly, most Ig domains present in RTKs and cell adhesion molecules belong to a distinct structural set known as the I set, with architecture intermediate between the V and C1 sets (2).MET, the RTK encoded by the c-met protoon...

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The structural characterization in crystals of three designed decapeptides containing a double d-segment at the C-terminus is described. The crystal structures of the peptides Boc-Leu-Aib-Val-Xxx-Leu-Aib-Val-(D)Ala-(D)Leu-Aib-OMe, (Xxx = Gly 2, (D)Ala 3, Aib 4) have been determined and compared with those reported earlier for peptide 1 (Xxx = Ala) and the all l analogue Boc-Leu-Aib-Val-Ala-Leu-Aib-Val-Ala-Leu-Aib-OMe, which yielded a perfect right-handed alpha-helical structure. Peptides 1 and 2 reveal a right-handed helical segment spanning residues 1 to 7, ending in a Schellman motif with (D)Ala(8) functioning as the terminating residue. Polypeptide chain reversal occurs at residue 9, a novel feature that appears to be the consequence of a C-H.O hydrogen bond between residue 4 C(alpha)H and residue 9 CO groups. The structures of peptides 3 and 4, which lack the pro R hydrogen at the C(alpha) atom of residue 4, are dramatically different. Peptide 3 adopts a right-handed helical conformation over the 1 to 7 segment. Residues 8 and 9 adopt alpha(L) conformations forming a C-terminus type I' beta-turn, corresponding to an incipient left-handed twist of the polypeptide chain. In peptide 4, helix termination occurs at Aib(6), with residues 6 to 9 forming a left-handed helix, resulting in a structure that accommodates direct fusion of two helical segments of opposite twist. Peptides 3 and 4 provide examples of chiral residues occurring in the less favored sense of helical twist; (D)Ala(4) in peptide 3 adopts an alpha(R) conformation, while (L)Val(7) in 4 adopts an alpha(L) conformation. The structural comparison of the decapeptides reported here provides evidence for the role of specific C-H.O hydrogen bonds in stabilizing chain reversals at helix termini, which may be relevant in aligning contiguous helical and strand segments in polypeptide structures.

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Summary The nuclear pore complex subunit TPR is found in at least five different oncogenic fusion kinases, including TPR-MET, yet how TPR fusions promote activation of kinases and their oncogenic activities remains poorly understood. Here we report the crystal structure of TPR(2-142), the MET fusion partner of oncogenic TPR-MET. TPR(2-142) contains a continuous 124-residue α-helix that forms an anti-parallel tetramer from two leucine zipper-containing parallel coiled coils. Remarkably, single mutations cause strikingly different conformations of the coiled coil, indicating its highly dynamic nature. We further show that fusion of TPR(2-142) to the MET intracellular domain strongly and selectively stabilizes the αG-helix of the MET kinase domain and mutations of only the TPR leucine zipper residues at the junction to MET, but not other leucine zipper residues, abolish kinase activation. Together, these results provide critical insight into the TPR structure and its ability to induce dimerization and activation of fusion kinases.