Limited proteolysis of recombinant human soluble interleukin-2 receptor. Identification of an interleukin-2 binding core.
Limited proteolysis of a recombinant, soluble form of the Tac protein, a human interleukin-2 receptor (rIL-2R), was performed using trypsin, Staphylococcus aureus V8 protease and proteinase K to study the structural requirements of interleukin-2 receptor (IL-2R) for interleukin-2 (IL-2) binding. Sensitive proteolytic sites were found to be clustered in the regions of the polypeptide encoded by exons 3, 5, and 6, with a few semi-sensitive sites located within the two homologous domains encoded by exons 2 and 4. A number of nicked and truncated rIL-2R species generated by proteolysis were assayed for IL-2 binding using recombinant IL-2 (rIL-2) affinity gel and then structurally characterized. The results demonstrated that only the species that consist of the regions encoded by exons 2 and 4, joined by five disulfide bonds, are capable of binding IL-2 and that the presence of semi-sensitive cleavage sites within the two homologous domains had no apparent effect on IL-2 binding. These results suggest that the pattern of the sensitive cleavage sites in rIL-2R is closely related to the structural requirements for IL-2 binding. Based on the experimental results, a highly symmetrical core structure of IL-2R with a total of 135 amino acid residues was identified. This is the smallest protein moiety so far known to be capable of binding IL-2.
Miedel
M C
MC
Department of Protein Biochemistry, Roche Research Center, Hoffmann-La Roche Inc., Nutley, New Jersey 07110.
Hulmes
J D
JD
Pan
Y C
YC
D000595:Amino Acid Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007376:Interleukin-2 / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010447:Peptide Hydrolases / Q000378:metabolism; D010449:Peptide Mapping; D011487:Protein Conformation; D015375:Receptors, Interleukin-2 / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D007376:Interleukin-2; D010446:Peptide Fragments; D015375:Receptors, Interleukin-2; D011994:Recombinant Proteins; D010447:Peptide Hydrolases