Structure-function studies on recombinant human macrophage colony-stimulating factor (M-CSF).
Human macrophage colony-stimulating factor (M-CSF) is a homodimeric cytokine that is a member of a structurally related family of hormones defined by an unusual up-up-down-down alpha-helical bundle. To identify regions on the surface of M-CSF that might interact with the M-CSF receptor, single and double amino acid substitutions were introduced into a truncated form of human M-CSF alpha by site-directed mutagenesis, and the homodimeric M-CSF analogs were purified and characterized. Certain substitutions in the region before and in helix A and in helix C decreased specific bioactivity and correlated with an approximately equivalent reduction in M-CSF receptor affinity. The most dramatic change was observed in an analog in which residues His-9 and His-15 were replaced with alanines, resulting in a 9,100-fold decrease in specific bioactivity. X-ray crystallographic analysis of this M-CSF alpha H9A,H15A analog at a resolution of 2.5 A revealed no significant changes in structure other than the expected new side chains at residues 9 and 15. Analogs containing only one of these two histidine substitutions exhibited a decrease in specific bioactivity of 6- and 1200-fold for the H9A and H15A mutations, respectively. It appears that selected amino acids in the NH2-terminal region of M-CSF and possibly portions of the surface formed by helices A and C are significantly involved in interactions with the M-CSF receptor.
Taylor
E W
EW
Department of Biological Therapeutics Research, Chiron Corporation, Emeryville, California 94608.
Fear
A L
AL
Bohm
A
A
Kim
S H
SH
Koths
K
K
D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D006801:Humans; D016173:Macrophage Colony-Stimulating Factor / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D016186:Receptor, Macrophage Colony-Stimulating Factor / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D011994:Recombinant Proteins; D016173:Macrophage Colony-Stimulating Factor; D016186:Receptor, Macrophage Colony-Stimulating Factor