Rational design of a mouse granulocyte macrophage-colony-stimulating factor receptor antagonist

Mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) proteins with substitutions at residues in the first alpha-helix were examined for biological activity and receptor binding properties. Substitution at the buried residue His15 affected both bioactivity and receptor binding. Of the four surface-exposed positions examined (Arg11, Lys14, Lys20, and Glu21) only substitutions at Glu21 impaired bioactivity. Proteins with charge reversal substitutions at this position were partial agonists and weak antagonists of native mGM-CSF action. All substitutions at Glu21 abrogated high affinity binding. Lys14 and Lys20 substitution proteins showed various receptor binding defects. Qualitative and quantitative measurement of these binding defects identified Lys14 as a residue that interacts specifically with the beta subunit of the mGM-CSF receptor, whereas Lys20 appeared to exist at the GM-R alpha-subunit/GM-R beta-subunit interface as substitutions at this position produce both high and low affinity binding losses. These determinations permitted the design of a more potent mGM-CSF antagonist.