Structure/function of HGF/SF and Met
Authors: Ermanno Gherardi
Journal: Cancer Research (2008)

SY28-03  The growth and motility factor Hepatocyte Growth Factor/Scatter Factor (HGF/SF) and its receptor, the tyrosine kinase encoded by the c-MET proto-oncogene, exert major roles in cancer invasion and metastasis and are key targets for therapy (1). Here I discuss our current understanding of the structure/function of these molecules, the implication for the mechanism of activation of MET and possible ways in which knowledge of the structure may help designing novel receptor antagonists for therapy.  Domain structure and proteolytic activation of HGF/SF  HGF/SF and its homologue HGFl/MSP constitute an unusual growth factor family because these proteins, although devoid of enzymatic activity, are structurally related to the complex proteinases of the clotting and fibrinolytic pathways. HGF/SF consists of six protein domains: an N-terminal domain (N), four copies of the kringle (K) domain and a C-terminal domain similar to the catalytic domain of serine proteinases (SP). This domain structure resembles closely that of the proteinase precursor plasminogen and, like plasminogen, HGF/SF is synthesised as a precursor protein (pro- or single-chain HGF/SF) and is subsequently converted to a two-chain form by cleavage of the linker connecting the K4 and SP domains. Single-chain HGF/SF binds MET but is unable to induce biological responses, whereas two-chain, mature HGF/SF is signalling-active. Three of the six domains of HGF/SF are directly involved in MET binding, namely the N, K1 and SP domains (2). The N and K1 domains, adjacent to each other, are responsible for the high-affinity binding site for MET whereas the SP domain provides a low-affinity binding site, both of which are required for MET activation by two-chain HGF/SF. Crystal structures are now available both for NK1 (a fragment of HGF/SF encompassing the N and K1 domains) (3, 4) and the SP domain (5). Using small-angle x-ray scattering (SAXS) and cryo-electron microscopy (cryo-EM) we have also gained direct clues into the structural basis of proteolytic activation of HGF/SF. Specifically, we have established that single-chain HGF/SF is a compact molecule in which the high- and low-affinity binding sites for MET are close to each other. In contrast, two-chain HGF/SF has an elongated, open structure in which the two receptor binding sites are more than 100A apart (6). This, and a local change in the binding site for MET of the SP domain resulting from proteolytic activation (5) suggest that the inability of single-chain HGF/SF to activate MET is due to monovalent binding through the high-affinity site alone whereas two-chain HGF/SF binds bivalently through the NK1 and SP binding sites (6).  Domain structure of MET and ligand-receptor complexes  Whereas the domain structure of HGF/SF was immediately apparent from sequence analysis, that of the large extracellular MET ectodomain (amino acids 25-932) had to be determined experimentally and was obtained by systematic deletion mutagenesis, ligand-binding studies and three-dimensional modelling. These studies led to the conclusion that the MET ectodomains consists of two main moieties: a globular head (amino acids 25-519) responsible for HGF/SF binding and with the fold of a 7-bladed beta-propeller and a flexible stalk (amino acids 562-932) consisting of four immunoglobulin-like (Ig) domains of an unusual structural set (7). The head and stalk structure are separated by a small cystine-rich domain (amino acids 520-561). There are now two crystal structures of fragments of the MET ectodomain in complex with ligands. The first of these comprises the SP domain of HGF/SF in complex with the beta-propeller and cystine-rich domain of MET (8). The second involves the beta-propeller, cystine-rich domain and the first two Ig domains of MET in complex with a fragment of an alternative MET ligand, the bacterial protein InlB of L monocytogenes (9). The latter structure shows that the primary binding site for InlB is not located in the beta-propeller domain, as is the case for the natural ligand HGF/SF but rather in the first Ig domain. It also suggests that the bacterial protein activates MET by a mechanism different from HGF/SF: whereas two-chain HGF/SF can form a complex with 2:2 stoichiometry with the MET beta-propeller, InlB activates MET by ligand oligomerisation mediated by cell surface heparan sulphate proteoglycans (9). Thus the two known MET ligands, the physiological ligand HGF/SF and the bacterial protein InlB, activate MET by a distinct structural mechanism.  Engineering HGF/SF fragments as receptor antagonists  The available crystal structures of NK1 have recently enabled the generation of engineered variants with antagonistic activity. NK1 is a partial receptor agonist and crystallises as a head to tail dimer with an extensive inter-protomeric interface resulting from contacts between the two short inter-domain linkers and reciprocal contacts between the N and K1 domains (3, 4). Systematic mutagenesis of the inter-protomeric interface has produced several mutants, such as single linker mutants Y124A and N127A or the doubke K1 mutant V140A:I142A, that bind the MET receptor with affinities comparable to wild type NK1 but fail to assemble a dimeric, signalling competent NK1-MET complex. These NK1 variants have no detectable agonistic activity on target cells and behave as bona fide receptor antagonists by blocking HGF/SF-induced cell growth migration in target cells (10, 11). Thus, they offer interesting prospects as therapeutics for human cancer. In summary, although high-resolution structures of full length HGF/SF in complex with the MET ectodomain have not yet been reported, the low-resolution SAXS and cryo-EM models and the partial crystal structures of fragments of HGF/SF and MET have produced a first picture of the structure/function of these two signalling proteins and the structural basis of HGF/SF and MET activation. They have also offered, at the same time, new approaches for the development of MET therapeutics.  References   1. Birchmeier, C., Birchmeier, W., Gherardi, E., et al. Met, metastasis, motility and more. Nat Rev Mol Cell Biol, 2003;4:915-25.  2. Holmes, O., Pillozzi, S., Deakin, J. A., et al. E. Insights into the structure/function of hepatocyte growth factor/scatter factor from studies with individual domains. J Mol Biol, 2007;367:395-408.  3. Chirgadze, D. Y., Hepple, J. P., Zhou, H., Byrd, R. A., Blundell, T. L., and Gherardi, E. Crystal structure of the NK1 fragment of HGF/SF suggests a novel mode for growth factor dimerization and receptor binding. Nat Struct Biol, 1999;6:72-79.  4. Ultsch, M., Lokker, N. A., Godowski, P. J., and de Vos, A. M. Crystal structure of the NK1 fragment of human hepatocyte growth factor at 2.0 A resolution. Structure, 1998;6:1383-1393.  5. Kirchhofer, D., Yao, X., Peek, M.,et al. Structural and functional basis of the serine protease-like hepatocyte growth factor beta-chain in Met binding and signaling. J Biol Chem, 2004;279: 9915-24.  6. Gherardi, E., Sandin, S., Petoukhov, M. V., et al Structural basis of hepatocyte growth factor/scatter factor and MET signalling. Proc Natl Acad Sci U S A, 2006;103:4046-51.  7. Gherardi, E., Youles, M. E., Miguel, R. N., et al Functional map and domain structure of MET, the product of the c-met protooncogene and receptor for hepatocyte growth factor/scatter factor. Proc Natl Acad Sci U S A, 2003;100:12039-44,.  8. Stamos, J., Lazarus, R. A., Yao, X., Kirchhofer, D., and Wiesmann, C. Crystal structure of the HGF beta-chain in complex with the Sema domain of the Met receptor. Embo J, 2004;23:2325-35.  9. Niemann, H., Jager, V., Butler, P.,et al. Structure of the human receptor tyrosine kinase met in complex with the listeria monocytogenes invasion protein InlB. Cell, 2007;130:235-46.  10. Tolbert, W. D., Daugherty, J., Gao, C.,et al. A mechanistic basis for converting a receptor tyrosine kinase agonist to an antagonist. Proc Natl Acad Sci U S A, 2007;104:14592-97.  11. Youles, E. M., Holmes, O., Pethoukhov, M. V.,et al. Engineering the NK1 fragment of Hepatocyte Growth Factor/Scatter Factor as a MET receptor antagonist. J Mol Biol, 2008;in press.