Structural basis for agonism and antagonism of hepatocyte growth factor

Abstract

Hepatocyte growth factor (HGF) is an activating ligand of the Met receptor tyrosine kinase, whose activity is essential for normal tissue development and organ regeneration but abnormal activation of Met has been implicated in growth, invasion, and metastasis of many types of solid tumors. HGF has two natural splice variants, NK1 and NK2, which contain the N-terminal domain (N) and the first kringle (K1) or the first two kringle domains of HGF. NK1, which is a Met agonist, forms a head-to-tail dimer complex in crystal structures and mutations in the NK1 dimer interface convert NK1 to a Met antagonist. In contrast, NK2 is a Met antagonist, capable of inhibiting HGF's activity in cell proliferation without clear mechanism. Here we report the crystal structure of NK2, which forms a "closed" monomeric conformation through interdomain interactions between the N-domain and the second kringle domain (K2). Mutations that were designed to open up the NK2 closed conformation by disrupting the N/K2 interface convert NK2 from a Met antagonist to an agonist. Remarkably, this mutated NK2 agonist can be converted back to an antagonist by a mutation that disrupts the NK1/NK1 dimer interface. These results reveal the molecular determinants that regulate the agonist/antagonist properties of HGF NK2 and provide critical insights into the dimerization mechanism that regulates the Met receptor activation by HGF.cancer metastasis | crystal structure | HGF agonist | HGF antagonist | Met receptor tyrosine kinase H epatocyte growth factor (HGF) and the Met tyrosine kinase receptor form a unique ligand-receptor signaling system where HGF is the only known endogenous ligand that activates Met. HGF is also known as a scattering factor, and its signaling through Met activation is involved in promoting cell proliferation, survival, migration, and branching in a variety of cell types with important roles in angiogenesis, wound repair, and cell migration during development (1, 2). Inappropriate expression of Met or HGF has been implicated in many cancers and is associated with an aggressive phenotype and poor prognosis (1, 3) (www.vai.org/met). Thus, inhibition of Met activity, either by small molecule kinase inhibitors or by protein-based HGF antagonists, has become an important and rational strategy for developing anticancer therapeutics.The Met extracellular domain (ECD), which encompasses approximately 900 amino acids (25-932), is cleaved into α and β chains by the furin protease between residues 307 and 308. The first 519 amino acids of Met form a 7-bladed β-propeller domain, called sema domain, with homology to the semaphorin and plexin protein families (4, 5). Following the sema domain are a cysteine-rich domain and four immunoglobulin-like domains. The sema domain of Met is necessary and sufficient for binding and activation of the receptor by HGF (6). It is believed that HGF induced Met activation is mediated through a formation of a 2∶2 complex where Met dimerization is primarily mediated by dimer formation of HGF (7,8).HGF is a disu...

Engineering hepatocyte growth factor fragments with high stability and activity as Met receptor agonists and antagonists

“…Heparin has been reported to enhance NK1-Met binding affinity (15,19,20), but here we report reasonably strong NK1-Met binding in the absence of heparin. This affinity is considerably stronger than previous measurements that used recombinant forms of the Met extracellular domain (20,25), suggesting that Met may adopt different conformations when expressed on the mammalian cell surface. It is also possible that the instability of wild-type NK1 has made accurate determination of its Met binding affinity challenging.…”

“…Restoration of the native NK1 homodimerization interface in the stability-enhanced mutant M2.2 D127N results in essentially identical agonistic activity to wild-type NK1, and disruption of this interface by the M2.2 D127A/K/and R mutants results in efficient receptor antagonism, further supporting the physiologic relevance of NK1-NK1 crystallographic interface (17,19,20). The improved NK1 mutants described here could support further development of other Met agonists that are comprised partially or wholly from the unstable wild-type NK1 fragment (33)(34)(35), and given their enhanced stability have potential to be readily incorporated into biomaterials for therapeutic applications (36).…”

“…NK2, another naturally occurring HGF splice variant, comprises the N-terminal and first two Kringle domains of HGF. NK2 can be transformed from an antagonist to an agonist by disrupting the NK1-K2 intraprotomer interaction and then can be turned back into an antagonist by introducing the same point mutations that block NK1 homodimerization (20).…”

Targeting the MET oncogene in cancer and metastases

“…Mutations that were designed to open up the NK2 closed conformation by disrupting the N/K2 interface convert NK2 into a MET agonist. [50]. It should be noted that, under certain conditions, these HGF fragments might behave as partial MET agonists, a property that may limit their therapeutic utility.…”

Structural Basis for the Binding Specificity of Human Recepteur d'Origine Nantais (RON) Receptor Tyrosine Kinase to Macrophage-stimulating Protein

“…By contrast, HGF␣ binds to the MET with an affinity higher than that of HGF␤ (37). In fact, splice variants of HGF, NK 1 and NK 2 , function as MET agonist and antagonist, respectively (38). To gain insights into the structural determinants of RON-MSP specificity, the crystal structure of the RON SPI 1 -MSP␤ complex was determined at 3.0 Å, and the binding interaction was characterized using analytical ultracentrifugation (AUC).…”