pH Dependence of structural stability of interleukin-2 and granulocyte colony-stimulating factor.
After a cytokine binds to its receptor on the cell surface (pH approximately 7), the complex is internalized into acidic endosomal compartments (pH approximately 5-6), where partially unfolded intermediates can form. The nature of these structural transitions was studied for wild-type interleukin-2 (IL-2) and wild-type granulocyte colony-stimulating factor (G-CSF). A noncoincidence of denaturation transitions in the secondary and tertiary structure of IL-2 and tertiary structural perturbations in G-CSF suggest the presence of an intermediate state for each, a common feature of this structural family of four-helical bundle proteins. Unexpectedly, both IL-2 and G-CSF display monotonic increases in stability as the pH is decreased from 7 to 4. We hypothesize that such cytokines with cell-based clearance mechanisms in vivo may have evolved to help stabilize endosomal complexes for sorting to lysosomal degradation. We show that mutants of both IL-2 and G-CSF have differential stabilities to their wild-type counterparts as a function of pH, and that these differences may explain the differences in ligand trafficking and depletion. Further understanding of the structural changes accompanying unfolding may help guide cytokine design with respect to ligand binding, endocytic trafficking, and, consequently, therapeutic efficacy.
Ricci
Margaret Speed
MS
Pharmaceutics Department, Amgen Inc., Thousand Oaks, California 91320, USA. mspeed@amgen.com
Sarkar
Casim A
CA
Fallon
Eric M
EM
Lauffenburger
Douglas A
DA
Brems
David N
DN
D004705:Endocytosis; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry*; D019791:Guanidine / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007376:Interleukin-2 / Q000737:chemistry*; D009154:Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D021381:Protein Transport
D007376:Interleukin-2; D016179:Granulocyte Colony-Stimulating Factor; D019791:Guanidine