Engineering and crystal structure of a monomeric FLT3 ligand variant

The overarching paradigm for the activation of class III and V receptor tyrosine kinases (RTKs) prescribes cytokine-mediated dimerization of the receptor ectodomains and homotypic receptor-receptor interactions. However, structural studies have shown that the hematopoietic receptor FLT3, a class III RTK, does not appear to engage in such receptor-receptor contacts, despite its efficient dimerization by dimeric FLT3 ligand (FL). As part of efforts to better understand the intricacies of FLT3 activation, we sought to engineer a monomeric FL. It was found that a Leu27Asp substitution at the dimer interface of the cytokine led to a stable monomeric cytokine (FLL27D) without abrogation of receptor binding. The crystal structure of FLL27D at 1.65 angstrom resolution revealed that the introduced point mutation led to shielding of the hydrophobic footprint of the dimerization interface in wild-type FL without affecting the conformation of the FLT3 binding site. Thus, FLL27D can serve as a monomeric FL variant to further interrogate the assembly mechanism of extracellular complexes of FLT3 in physiology and disease.