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. Study holds ProTherm entries: 16246, 16247, 16248, 16249, 16250, 16251, 16252, 16253, 16254, 16255, 16256, 16257, 16258, 16259, 16260, 16261, 16262, 16263, 16264, 16265 Extra Details: protein folding; protein stability; equilibrium denaturation; folding intermediates;,endocytic trafficking; IL-2; G-CSF
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:47 p.m.
|Number of data points||54|
|Proteins||Granulocyte colony-stimulating factor ; Interleukin-2 ; Granulocyte-macrophage colony-stimulating factor ; Interleukin-2|
|Assays/Quantities/Protocols||Experimental Assay: Cm prot_conc:0.050 mg/mL, pH:7.0 ; Experimental Assay: dG_H2O prot_conc:0.050 mg/mL, pH:7.0 ; Experimental Assay: Cm pH:4.0, prot_conc:0.050 mg/mL ; Experimental Assay: dG_H2O pH:4.0, prot_conc:0.050 mg/mL ; Experimental Assay: Cm prot_conc:0.025-0.050 mg/mL, pH:7.0 ; Experimental Assay: dG_H2O prot_conc:0.025-0.050 mg/mL, pH:7.0 ; Experimental Assay: Cm prot_conc:0.025-0.050 mg/mL, pH:4.0 ; Experimental Assay: dG_H2O prot_conc:0.025-0.050 mg/mL, pH:4.0 ; Derived Quantity: ddG_H2O prot_conc:0.050 mg/mL, pH:7.0 ; Derived Quantity: ddG_H2O pH:4.0, prot_conc:0.050 mg/mL ; Derived Quantity: ddG_H2O prot_conc:0.025-0.050 mg/mL, pH:7.0 ; Derived Quantity: ddG_H2O prot_conc:0.025-0.050 mg/mL, pH:4.0|
|Libraries||Mutations for sequence TPLGPASSLPQSFLLKCLEQVRKIQGDGAALQEKLCATYKLCHPEELVLLGHSLGIPWAPLSSCPSQALQLAGCLSQLHSGLFLYQGLLQALEGISPELGPTLDTLQLDVADFATTIWQQMEELGMAPALQPTQGAMPAFASAFQRRAGGVLVASHLQSFLEVSYRVLRHLAQP ; Mutations for sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT|