MULTIPLE CONFORMATIONS OF A HUMAN INTERLEUKIN-3 VARIANT

Interleukin-3 (IL-3) is a cytokine that stimulates the proliferation a nd differentiation of hematopoietic cells. The hyperactive hIL-3 varia nt 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 hypothe sis that proline cis-trans isomerization is the source of the observed conformational heterogeneity, as well as to evaluate the effect of pr olyl peptide bond configuration on biological activity. NMR spectra of four single proline-to-alanine mutants (P30A, P31A, P33A, and P37A) r etain 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 confor mations in SC-55494 correspond to cis and trans isomers of either or b oth of the R29-P30 and P30-P31 peptide bonds. All six mutants had some what lower cell proliferative activity than SC-55494, with relative ac tivities ranging from 40 to 80%. The P37A mutant has a binding affinit y to the low-affinity IL-3 receptor alpha-subunit statistically equiva lent to SC-55494, while P30A, P31A, and P33A each had about two-fold d ecreases, and P30A/P31A and P30A/P31A/P33A/P37A had fourfold decreases . These findings suggest an important role for the cis configuration o f either or both of the R29-P30 and P30-P31 peptide bonds in IL-3 for optimal interaction with the receptor alpha-subunit.