Hammond M E, Shyamala V, Siani M A, Gallegos C A, Feucht P H, Abbott J, Lapointe G R, Moghadam M, Khoja H, Zakel J, Tekamp-Olson P
Chiron Corporation, Emeryville, California 94608, USA.
J Biol Chem. 1996 Apr 5;271(14):8228-35. doi: 10.1074/jbc.271.14.8228.
To determine the regions of interleukin-8 (IL-8) that allow high affinity and interleukin-8 receptor type 1 (IL8R1)-specific binding of chemokines, we produced chimeric proteins containing structural domains from IL-8, which binds to both IL8R1 and interleukin-8 receptor type 2 (IL8R2) with high affinity, and from GRO gamma, which does not bind to IL8R1 and binds to IL8R2 with reduced affinity. Receptor binding activity was tested by competition of 125I-IL-8 binding to recombinant IL8R1 and IL8R2 cell lines. Substitution into IL-8 of the GRO gamma sequences corresponding to either the amino-terminal loop (amino acids 1-18) or the first beta-sheet (amino acids 18-32) reduced binding to both IL8R1 and IL8R2. The third beta-sheet of IL-8 (amino acids 46-53) was required for binding to IL8R1 but not IL8R2. Exchanges of the second beta-sheet (amino acids 32-46) or the carboxyl-terminal alpha-helix (amino acids 53-72) had no significant effect. When IL-8 sequences were substituted into GRO gamma, a single domain containing the second beta-sheet of IL-8 (amino acids 18-32) was sufficient to confer high affinity binding for both IL8R1 and IL8R2. The amino-terminal loop (amino acids 1-18) and the third beta-sheet (amino acids 46-53) of IL-8 had little effect when substituted individually but showed increased binding to both receptors when substituted in combination. Individual amino acid substitutions were made at positions where IL-8 and GRO gamma sequences differ within the regions of residues 11-21 and 46-53. IL-8 mutations L49A or L49F selectively inhibited binding to IL8R1. Mutations Y13L and F21N enhanced binding to IL8R1 with little effect on IL8R2. A combined mutation Y13L/S14Q selectively decreased binding to IL8R2. Residues Tyr13, Ser14, Phe21, and Lys49 are clustered in and around a surface-accessible hydrophobic pocket on IL-8 that is physically distant from the previously identified ELR binding sequence. A homology model of GRO gamma, constructed from the known structure of IL-8 by refinement calculations, indicated that access to the hydrophobic pocket was effectively abolished in GRO gamma. These studies suggest that the surface hydrophobic pocket and/or adjacent residues participate in IL-8 receptor recognition for both IL8R1 and IL8R2 and that the hydrophobic pocket itself may be essential for IL8R1 binding. Thus this region contains a second site for IL-8 receptor recognition that, in combination with the Glu4-Leu5-Arg6 region, can modulate receptor binding affinity and IL8R1 specificity.
为了确定白细胞介素-8（IL-8）中允许趋化因子与白细胞介素-8受体1型（IL8R1）进行高亲和力且特异性结合的区域，我们制备了嵌合蛋白，其包含来自IL-8的结构域（IL-8能与IL8R1和白细胞介素-8受体2型（IL8R2）均以高亲和力结合）以及来自GROγ的结构域（GROγ不与IL8R1结合，与IL8R2的结合亲和力较低）。通过125I-IL-8与重组IL8R1和IL8R2细胞系结合的竞争试验来检测受体结合活性。将与氨基末端环（氨基酸1 - 18）或第一个β折叠（氨基酸18 - 32）相对应的GROγ序列替换到IL-8中，会降低其与IL8R1和IL8R2的结合。IL-8的第三个β折叠（氨基酸46 - 53）是与IL8R1结合所必需的，但与IL8R2结合不需要。第二个β折叠（氨基酸32 - 46）或羧基末端α螺旋（氨基酸53 - 72）的交换没有显著影响。当将IL-8序列替换到GROγ中时，一个包含IL-8第二个β折叠（氨基酸18 - 32）的单一结构域足以赋予其与IL8R1和IL8R2的高亲和力结合。IL-8的氨基末端环（氨基酸1 - 18）和第三个β折叠（氨基酸46 - 53）单独替换时影响较小，但组合替换时会增加与两种受体的结合。在氨基酸11 - 21和46 - 53区域内，对IL-8和GROγ序列不同的位置进行了单个氨基酸替换。IL-8突变L49A或L49F选择性抑制与IL8R1的结合。突变Y13L和F21N增强了与IL8R1的结合，对IL8R2影响较小。组合突变Y13L/S14Q选择性降低与IL8R2的结合。酪氨酸13、丝氨酸14、苯丙氨酸21和赖氨酸49聚集在IL-8表面可及的疏水口袋及其周围，该口袋在空间上远离先前确定的ELR结合序列。通过精细计算从IL-8的已知结构构建的GROγ同源模型表明，GROγ中该疏水口袋的可及性被有效消除。这些研究表明，表面疏水口袋和/或相邻残基参与了IL-8对IL8R1和IL8R2的受体识别，并且疏水口袋本身可能是与IL8R1结合所必需的。因此，该区域包含IL-8受体识别的第二个位点，与Glu4 - Leu5 - Arg6区域结合可调节受体结合亲和力和IL8R1特异性。
