Lowman H B, Slagle P H, DeForge L E, Wirth C M, Gillece-Castro B L, Bourell J H, Fairbrother W J
Department of Protein Engineering, Genentech, Inc., South San Francisco, California 94080, USA.
J Biol Chem. 1996 Jun 14;271(24):14344-52. doi: 10.1074/jbc.271.24.14344.
Interleukin-8 (IL-8), a CXC chemokine, is known to bring about chemotaxis and activation of neutrophils through high affinity binding to at least two distinct receptors, receptor-A and receptor-B. The IL-8 homolog melanoma growth stimulating activity (MGSA) is also active toward neutrophils. In contrast to IL-8, MGSA binds receptor-B with high affinity and binds receptor-A with approximately 400-fold lower affinity. Using the structure of IL-8 (Clore et al.(1990) Biochemistry, 29, 1689-1696; Baldwin et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 502-506) and the NMR-determined structure of MGSA (Fairbrother et al. (1994) J. Mol. Biol. 242, 252-270), we designed variants of both IL-8 and MGSA to investigate the basis of specificity for binding of these chemokines to the IL-8 receptors. The most outstanding structural difference between IL-8 and MGSA lies in the loop preceding the first beta-strand. When the corresponding (shorter) loop from MGSA was swapped into IL-8, both receptor-A and receptor-B binding affinities were significantly (>300-fold) reduced. However, with additional mutations that affect packing interactions, an IL-8 variant specific for receptor-B binding was produced. Conversely, when the same loop from IL-8 was swapped into MGSA, receptor-B binding was maintained with only a approximately 30-fold reduction in receptor-A affinity. Again, mutations affecting packing of the loop yielded a MGSA variant with high affinity for both receptors, like IL-8. Finally, we show, through point mutations in a monomeric IL-8 framework, that individual side chain substitutions can affect receptor specificity.
白细胞介素-8（IL-8）是一种CXC趋化因子，已知它通过与至少两种不同的受体（受体-A和受体-B）高亲和力结合来引起中性粒细胞的趋化作用和激活。IL-8的同系物黑色素瘤生长刺激活性（MGSA）对中性粒细胞也有活性。与IL-8相反，MGSA与受体-B高亲和力结合，与受体-A的结合亲和力约低400倍。利用IL-8的结构（Clore等人，（1990年）《生物化学》，29卷，1689 - 1696页；Baldwin等人，（1991年）《美国国家科学院院刊》88卷，502 - 506页）以及MGSA的核磁共振确定结构（Fairbrother等人，（1994年）《分子生物学杂志》242卷，252 - 270页），我们设计了IL-8和MGSA的变体，以研究这些趋化因子与IL-8受体结合特异性的基础。IL-8和MGSA之间最显著的结构差异在于第一个β链之前的环。当将MGSA相应的（较短）环替换到IL-8中时，受体-A和受体-B的结合亲和力都显著降低（>300倍）。然而，通过影响堆积相互作用的额外突变，产生了一种对受体-B结合具有特异性的IL-8变体。相反，当将IL-8的同一环替换到MGSA中时，受体-B的结合得以维持，而受体-A的亲和力仅降低约30倍。同样，影响环堆积的突变产生了一种对两种受体都具有高亲和力的MGSA变体，类似于IL-8。最后，我们通过在单体IL-8框架中的点突变表明，单个侧链取代可以影响受体特异性。
