Structural and functional characterization of the MET receptor with surrogate ligands

• Applicant: Professor Dr. Hartmut Niemann
• Subject Area: Structural Biology ; Biochemistry
• Term: from 2016 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 288981880

For some single-pass transmembrane receptors like the Epo receptor or various receptor tyrosine kinases (RTKs), mere dimerization of their ectodomain may not suffice to efficiently activate intracellular signaling pathways. Alternatively, a particular arrangement of the two receptors ectodomains may be important, e.g. resulting in a relative rotation of the two transmembrane helices in the dimer. For some RTKs a weak dimerization contact of membrane-proximal receptor domains is essential in addition to the ligand-mediated dimerization. Such receptor-mediated contacts in the ectodomain ensure the correct arrangement of the two transmembrane helices.These issues are also relevant for the RTK MET. So far, the complex with the Listeria monocytogenes invasion protein InlB is the only available structure of MET in a signaling-active complex with a ligand. There is no structure of a MET dimer with its endogenous ligand hepatocyte growth factor (HGF). Many questions with regard to the structural basis of MET activation remain unanswered. For example, the structure of several membrane-proximal Ig-like domains in the MET ectodomain is unknown. Likewise, it is unclear, whether the four Ig-like domains contribute to MET activation through receptor-mediated dimerization contacts or whether they are just spacers, as has been suggested.Artificial MET ligands can help to resolve these questions. Prof. Gherardi and Prof. Plückthun have kindly provided us with two highly agonistic sheep anti-MET antibodies and with several designed ankyrin repeat protein (DARPins) directed against MET, respectively. We plan to use non-agonistic ligands (monomeric Fab fragments and DARPins) as crystallization chaperones to facilitate structure determination of the complete MET ectodomain, including all membrane-proximal Ig-like domains. We also have generated diabodies (dimeric, bivalent single-chain Fv fragments) derived from the sheep antibodies. These diabodies are as potent agonists as the intact antibodies, but are less flexible and hence more likely to crystallize. The structure of a diabody in complex with two MET molecules could clarify, whether the arrangement of two ectodomains in a signaling-active MET dimer matters or not. As a reference, we will use the signaling-active 2:2 complex of MET and L. monocytogenes InlB. In the future, structures of MET in complex with HGF or its splice variant NK1 may also become available for comparison.In yet unpublished experiments, we have characterized a sheep anti-MET diabody in solution using light scattering and small angle X-ray scattering. We also obtained first crystals of an isolated sheep Fab and a diabody. In addition we studied complex formation between the artificial binders and MET by gel filtration to identify the best candidates for co-crystallization.Detailed structural knowledge of the artificial MET ligands and their complexes with MET will contribute to developing them further for therapeutic purposes.

• DFG Programme: Research Grants