STRUCTURE-FUNCTION ANALYSIS OF FLT3 LIGAND-FLT3 RECEPTOR INTERACTIONSUSING A RAPID FUNCTIONAL SCREEN

FLT3 ligand (FLT3L) stimulates primitive hematopoietic cells by bindin g to and activating the FLT3 receptor (FLT3R), We carried out a struct ure-activity study of human FLT3L in order to define the residues invo lved in receptor binding. We developed a rapid method to screen random ly mutagenized FLT3L using a FLT3R-Fc fusion protein to probe the rela tive binding activities of mutated ligand, Approximately 60,000 potent ial mutants were screened, and the DNA from 59 clones was sequenced. T hirty-one single amino acid substitutions at 24 positions of FLT3L eit her enhanced or reduced activity in receptor binding and cell prolifer ation assays. Eleven representative proteins were purified and analyze d for receptor affinity, specific activity, and physical properties. R eceptor affinity and bioactivity were highly correlated. FLT3L affinit y for receptor improved when four individual mutations that enhance FL T3L receptor affinity were combined in a single molecule. A model of F LT3L three-dimensional structure was generated based on sequence align ment and x-ray structure of macrophage colony-stimulating factor. Most residues implicated in receptor binding are widely dispersed in the p rimary structure of FLT3L, yet they localize to a surface patch in the tertiary model. A mutation that maps to and is predicted to disrupt t he proposed dimerization interface between FLT3L monomers exhibits a S tokes radius that is concentration-dependent, suggesting that this mut ation disrupts the FLT3L dimer.