Crystal structures of NK1-heparin complexes reveal the basis for NK1 activity and enable engineering of potent agonists of the MET receptor

Lietha, Daniel; Chirgadze, D.Y.; Mulloy, M.; Blundell, Tom L.; Gherardi, Ermanno

doi:10.1107/s0108767302096319

Cited by 24 publications

(40 citation statements)

References 58 publications

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“…Some of these mutations are similar to those made by Lietha et al (18). These NK1 mutants were made with the additional R134G mutation to aid in thioredoxin tag removal and purification (8).…”

Section: Resultsmentioning

confidence: 99%

“…Binding of NK1 and NK2 to Met, as determined by competition of the binding of biotinylated NK1 to Met in AlphaScreen Assays, in the absence (A) and presence (B) of heparin. Both NK1 and NK2 contain the K132E and R134E mutations that were designated as 1K1 (18). (C and D).…”

Section: Resultsmentioning

confidence: 99%

“…The first, NK1, acts as a Met agonist, but requires the presence of heparan sulfate for full activity (17)(18)(19). NK1 contains a portion of the full HGF α-chain that includes the N terminus and the first kringle domains.…”

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confidence: 99%

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Structural basis for agonism and antagonism of hepatocyte growth factor

Tolbert¹,

Daugherty-Holtrop²,

Gherardi

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Structural basis for agonism and antagonism of hepatocyte growth factor

Tolbert¹,

Daugherty-Holtrop²,

Gherardi

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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“…The mutant 1K1 carries two reverse charge mutations at K132 and R134 (1K1: K132E:R134E) that disrupt heparin binding to the kringle domain ( Fig. 1) and displays increased signaling activity on several cell types compared to wild type (16). The higher biological activity of 1K1 over NK1 is due to two reasons: Firstly, as a result of the loss of the heparin-binding site in K1, heparin interacts solely with the primary binding site in the N domain and causes adjacent 1K1 dimers to bind Hepatocyte growth factor receptor (MNNG (N-Methyl-N'-nitro-Nnitrosoguanidine) HOS Tranforming gene) (MET) on the same plane (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In the presence of heparan sulfate or heparin, NK1 behaves as a partial receptor agonist (14). In order to develop 1K1, a rational protein engineering approach was employed based on structural and functional studies of NK1 (15), NK1-heparin complexes (16), and individual N and K1 domains (17). NK1 binds heparin through two distinct sites: a high-affinity site located in the N domain and formed by the side chains of R73, K60, T61, R76, K62, and K58 and main chain atoms of T61, K63, and G79 (18) and a low-affinity site in the K1 domain comprising the side chains of K132, R134, K170, and R181.…”

Section: Resultsmentioning

confidence: 99%

Coupling growth-factor engineering with nanotechnology for therapeutic angiogenesis

Roy

Soni

Harfouche

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Therapeutic angiogenesis is an emerging paradigm for the management of ischemic pathologies. Proangiogenic Therapy is limited, however, by the current inability to deliver angiogenic factors in a sustained manner at the site of pathology. In this study, we investigated a unique nonglycosylated active fragment of hepatocyte growth factor/scatter factor, 1K1, which acts as a potent angiogenic agent in vitro and in a zebrafish embryo and a murine matrigel implant model. Furthermore, we demonstrate that nanoformulating 1K1 for sustained release temporally alters downstream signaling through the mitogen activated protein kinase pathway, and amplifies the angiogenic outcome. Merging protein engineering and nanotechnology offers exciting possibilities for the treatment of ischemic disease, and furthermore allows the selective targeting of downstream signaling pathways, which translates into discrete phenotypes.N eovascularization in the adult holds the potential for ameliorating ischemic disease, which is the leading cause of morbidity and mortality in the United States (1). Ferrara and Kerbel recently articulated that "therapeutic angiogenesis" is an emerging and exciting frontier of cardiovascular medicine (2). However, while early preclinical studies and phase I clinical trials exhibited promising results with recombinant proteins or gene therapy, more rigorous phase II and III studies have reported conflicting outcomes (3-6). A recent review of these clinical trials highlighted vector "washout" leading to inadequate duration of exposure to the angiogenic agent as a key factor that may have impaired the effectiveness of therapy (7). Indeed, it is now established that sustained activation of downstream signal pathways promotes cellular processes critical for angiogenesis (8). We rationalized that harnessing a nanotechnology-based approach can enable a temporal control over the presentation of the angiogenic factors to the cellular target, thereby altering the activation of downstream signal transduction.While nanoformulation of angiogenic proteins is an attractive strategy, the synthesis and instability of these large complex glycosylated structures pose a significant barrier (9). Hepatocyte growth factor/scatter factor (HGF/SF) is a potent angiogenic agent (10), signaling through the Met receptor and downstream MAPK and the PI3K pathways (11-12). Interestingly, the Met receptor is upregulated during hypoxia, suggesting that the ischemic tissue will be primed for activation by HGF/SF (13). These results indicate that HGF/SF could emerge as an attractive therapy for angiogenesis. However, HGF/SF is a heparin-binding protein with a complex multidomain structure consisting of an N-terminal (N) domain, four copies of the kringle domain (K1 to K4) and a C-terminal domain homologous to that of serine proteineases (SP) (Fig. 1A) (14). Biologically active HGF/SF is produced by proteolytic cleavage of the linker connecting the fourth kringle and the SP domain yielding a two-chain (α/β)-heterodimer, in which both the α-and the...

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Dissociation of c‐Met phosphotyrosine sites in human cells in response to mouse hepatocyte growth factor but not human hepatocyte growth factor: the possible roles of different amino acids in different species

Ikebuchi

Oka

Mizuno

et al. 2012

Cell Biochemistry & Function

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Hepatocyte growth factor (HGF) is essential for embryogenesis, tissue regeneration and tumour malignancy through the activation of its receptor, c-Met. We previously demonstrated that HGF α-chain hairpin-loop, K1 domain and β-chain are required for c-Met signalling. The sequential phosphorylation of tyrosine residues, from c-Met kinase domain to multidocking regions, is required for HGF-signalling transduction. Herein, we provide evidence that the disconcerted activation of c-Met tyrosine regions fails to induce biological functions. When human cells were incubated with 'mouse HGF', kinase domain activation (i.e. phospho-Tyr-1230/34/35) became evident, but the multidocking site (i.e. Tyr-1349) was not phosphorylated, resulting in unsuccessful induction of migration and mitogenesis. The binding ability of mouse HGF α-chain, or of β-chain, to human c-Met was lower than that of human HGF, as evidenced by HGF-chimera assay. Notably, only four amino acid positions in HGF α-chain hairpin-loop and K1 domain and six positions in β-chain differed between human HGF and mouse HGF. The human-specific amino acids (such as Gln-95 in hairpin-loop, Arg-134 in K1 domain and Cys-561 in β-chain) may be important for accurate c-Met assembly and signalling transduction.

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Crystal structures of NK1-heparin complexes reveal the basis for NK1 activity and enable engineering of potent agonists of the MET receptor

Cited by 24 publications

References 58 publications

Structural basis for agonism and antagonism of hepatocyte growth factor

Structural basis for agonism and antagonism of hepatocyte growth factor

Coupling growth-factor engineering with nanotechnology for therapeutic angiogenesis

Dissociation of c‐Met phosphotyrosine sites in human cells in response to mouse hepatocyte growth factor but not human hepatocyte growth factor: the possible roles of different amino acids in different species

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