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Multiple conformations of a human interleukin‐3 variant
Author(s) -
Feng Yiqing,
Hood William F.,
Forgey Robert W.,
Abegg Ann L.,
Caparon Marie H.,
Thiele Barrett R.,
Leimgruber Richard M.,
Mcwherter Charles A.
Publication year - 1997
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560060821
Subject(s) - mutant , alanine , receptor , chemistry , proline , peptide , stereochemistry , isomerization , alanine scanning , protein subunit , peptide bond , microbiology and biotechnology , biology , biochemistry , amino acid , mutagenesis , gene , catalysis
Abstract Interleukin‐3 (IL‐3) is a cytokine that stimulates the proliferation and differentiation of hematopoietic cells. The hyperactive hIL‐3 variant SC‐55494 was shown to have at least two major conformations by high‐resolution NMR spectroscopy. Mutants of SC‐55494 were constructed in which alanine was substituted for proline in order to test the hypothesis that proline cis‐trans isomerization is the source of the observed conformational heterogeneity, as well as to evaluate the effect of prolyl peptide bond configuration on biological activity. NMR spectra of four single proline‐to‐alanine mutants (P30A, P31A, P33A, and P37A) retain doubled resonances, while spectra of the double mutant P30A/P31A and the quadruple mutant P30A/P31A/P33A/ P37A are substantially free of heterogeneity. These observations suggest that the two major conformations in SC‐55494 correspond to cis and trans isomers of either or both of the R29‐P30 and P30‐P31 peptide bonds. All six mutants had somewhat lower cell proliferative activity than SC‐55494, with relative activities ranging from 40 to 80%. The P37A mutant has a binding affinity to the low‐affinity IL‐3 receptor α‐subunit statistically equivalent to SC‐55494, while P30A, P31A, and P33A each had about two‐fold decreases, and P30A/P31A and P30A/P31A/P33A/P37A had four‐fold decreases. These findings suggest an important role for the cis configuration of either or both of the R29‐P30 and P30‐P31 peptide bonds in IL‐3 for optimal interaction with the receptor α‐subunit.