Feng Y, Hood W F, Forgey R W, Abegg A L, Caparon M H, Thiele B R, Leimgruber R M, McWherter C A
G.D. Searle and Company, St. Louis, Missouri 63198, USA.
Protein Sci. 1997 Aug;6(8):1777-82. doi: 10.1002/pro.5560060821.
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-alamine 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 alpha-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 alpha-subunit.
白细胞介素-3（IL-3）是一种刺激造血细胞增殖和分化的细胞因子。通过高分辨率核磁共振光谱显示，活性过高的人IL-3变体SC-55494至少有两种主要构象。构建了SC-55494的突变体，其中用丙氨酸取代脯氨酸，以检验脯氨酸顺反异构化是观察到的构象异质性来源的假设，同时评估脯氨酰肽键构型对生物活性的影响。四个单脯氨酸到丙氨酸突变体（P30A、P31A、P33A和P37A）的核磁共振光谱保留了双峰，而双突变体P30A/P31A和四突变体P30A/P31A/P33A/P37A的光谱基本没有异质性。这些观察结果表明，SC-55494中的两种主要构象对应于R29-P30和P30-P31肽键中一个或两个的顺式和反式异构体。所有六个突变体的细胞增殖活性均略低于SC-55494，相对活性范围为40%至80%。P37A突变体与低亲和力IL-3受体α亚基的结合亲和力在统计学上与SC-55494相当，而P30A、P31A和P33A的结合亲和力各自降低了约两倍，P30A/P31A和P30A/P31A/P33A/P37A降低了四倍。这些发现表明，IL-3中R29-P30和P30-P31肽键中一个或两个的顺式构型对于与受体α亚基的最佳相互作用具有重要作用。
