PROTEINS: Structure, Function, and Genetics 26304-313 (1996) Refined Crystal Structure and Mutagenesis of Human Granulocyte-Macrophage Colony-Stimulating Factor

ABSTRACT The crystal structure of re-combinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) has been refined against data extending to a resolution of-2.4 A along a * and-1.9 A along b * and c*. Anisotropic scale factors of B,, =-20.8 A’, B,, = 7.4 A’, B, = 13.3 A ’ corrected for the more rapid fall of diffraction in the a * direction. The anisotropy correlates with the weak crystal packing interactions along the a axis. In addi-tion to apolar side chains in the protein core, there are 10 buried hydrogen bonding residues. Those residues involved in intramolecular hy-drogen bonding to main chain atoms are better conserved than those hydrogen bonding to other side chain atoms; 24 solvation sites are observed at equivalent positions in the two mol-ecules in the asymmetric unit, and the strongest among these are located in clefts between sec-ondary structural elements. No buried water sites are seen. Two surface clusters of hydro-phobic side chains are located near the ex-pected receptor binding regions. Mutagenesis of 11 residues on the helix Nhelix C face con-firms the importance of Glu-21 and shows that Gly-75 and Gln-86, located on helix C, each cause a greater than fourfold drop in activity. Glu-21 and Gly-75, but not Gln-86, are structur-ally equivalent to residues involved in the growth hormone binding to its receptor. 0 1996 Wiley-Liss, Inc