Protein engineering of MET antagonists
This work aims to engineer the NK1 fragment of HGF/SF (amino acids 31-206) as a receptor antagonist. NK1 is an attractive building block for a MET antagonist because it binds the MET receptor with good affinity and can be expressed in high yields in yeast cells in striking contrast with NK4, full length HGF/SF or the MET ectodomain.
The generation of an NK1-based MET antagonist requires two steps: in the first the residual agonistic activity of NK1 will be removed, in the second the binding affinity of the protein antagonist will be increased. These two steps are outlined below.
We have produced several crystal structures of NK1 in different crystallographic environments. These structures consistently showed an NK1 dimer in a head-to-tail arrangement, in which residues which contact the MET receptor are located on the exposed face of each protomer while the opposite face forms an extensive dimer interface with contacts between the two inter-domain linkers (L-L) and the N and K1 domains of the two protomers (N-K1). We have introduced point mutations at the interprotomeric interface and have shown that some of these mutants behave as bona fide receptor antagonists.
These proteins, therefore, are now ready for the subsequent stage of the work, namely engineering the affinity of NK1 for MET either by structure-based design or by random mutagenesis and selection.