Verdino Petra, Aldag Caroline, Hilvert Donald, Wilson Ian A
Department of Molecular Biology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Proc Natl Acad Sci U S A. 2008 Aug 19;105(33):11725-30. doi: 10.1073/pnas.0801783105. Epub 2008 Aug 8.
Molecular recognition by the adaptive immune system relies on specific high-affinity antibody receptors that are generated from a restricted set of starting sequences through homologous recombination and somatic mutation. The steroid binding antibody DB3 and the catalytic Diels-Alderase antibody 1E9 derive from the same germ line sequences but exhibit very distinct specificities and functions. However, mutation of only two of the 36 sequence differences in the variable domains, Leu(H47)Trp and Arg(H100)Trp, converts 1E9 into a high-affinity steroid receptor with a ligand recognition profile similar to DB3. To understand how these changes switch binding specificity and function, we determined the crystal structures of the 1E9 Leu(H47)Trp/Arg(H100)Trp double mutant (1E9dm) as an unliganded Fab at 2.05 A resolution and in complex with two configurationally distinct steroids at 2.40 and 2.85 A. Surprisingly, despite the functional mimicry of DB3, 1E9dm employs a distinct steroid binding mechanism. Extensive structural rearrangements occur in the combining site, where residue H47 acts as a specificity switch and H100 adapts to different ligands. Unlike DB3, 1E9dm does not use alternative binding pockets or different sets of hydrogen-bonding interactions to bind configurationally distinct steroids. Rather, the different steroids are inserted more deeply into the 1E9dm combining site, creating more hydrophobic contacts that energetically compensate for the lack of hydrogen bonds. These findings demonstrate how subtle mutations within an existing molecular scaffold can dramatically modulate the function of immune receptors by inducing unanticipated, but compensating, mechanisms of ligand interaction.
适应性免疫系统的分子识别依赖于特定的高亲和力抗体受体，这些受体通过同源重组和体细胞突变从一组有限的起始序列产生。类固醇结合抗体DB3和催化狄尔斯-阿尔德酶抗体1E9源自相同的种系序列，但表现出非常不同的特异性和功能。然而，可变区36个序列差异中只有两个发生突变，即Leu(H47)Trp和Arg(H100)Trp，就能将1E9转化为具有与DB3相似配体识别谱的高亲和力类固醇受体。为了理解这些变化如何改变结合特异性和功能，我们确定了1E9 Leu(H47)Trp/Arg(H100)Trp双突变体（1E9dm）未结合配体的Fab晶体结构，分辨率为2.05 Å，以及与两种构型不同的类固醇形成复合物时的晶体结构，分辨率分别为2.40 Å和2.85 Å。令人惊讶的是，尽管1E9dm在功能上模拟了DB3，但它采用了不同的类固醇结合机制。结合位点发生了广泛的结构重排，其中H47残基作为特异性开关，H100适应不同的配体。与DB3不同，1E9dm不使用替代结合口袋或不同的氢键相互作用组来结合构型不同的类固醇。相反，不同的类固醇更深地插入到1E9dm结合位点，形成更多的疏水接触，在能量上补偿了氢键的缺乏。这些发现证明了在现有的分子支架内的细微突变如何通过诱导意想不到但具有补偿作用的配体相互作用机制来显著调节免疫受体的功能。
