The structural basis for DNA binding by an anti-DNA autoantibody
The structural basis for DNA binding by an anti-DNA autoantibody
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DOI：
10.1016/s0161-5890(98)00095-9
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发表时间：
1998-12-01
影响因子：
3.6
通讯作者：
Stollar, BD
中科院分区：
文献类型：
--
作者：
Jang, YJ;Sanford, D;Stollar, BD
我们已经使用了单点和多点定向诱变，和分子建模，以确定关键残基的DNA结合位点的单克隆抗体2C 10，IgG抗dsDNA自身抗体从MRL/lpr狼疮小鼠。同时替换CDR 3 H中的四个Arg残基消除了结合活性。除了一个例外，替换这些Arg残基中的任何一个将活性降低至未突变的scFv活性的20-50%。Arg到Asp的替换比Arg到Ala的替换具有稍大的效果。在一个例外的情况下，用Ala取代Arg 99实际上增加了DNA结合五倍，而用Asp取代几乎没有影响。在CDR 1H中Phe 32和Asn 35突变为Ala降低了DNA结合，而用Ala替换Arg 31具有可忽略的影响。在CDR 1 L中的一簇Asp残基中的任何一个的Ala取代使DNA结合增加三至六倍，证实了先前的发现，即MAb 2C 10的L链不利于DNA结合。L-链确实参与形成抗原结合的选择性，并且CDR 3L残基Asp 92或Asn 93的突变引起DNA结合活性的降低。与分子模型一致的定向诱变表明：几个CDR氨基酸有助于DNA结合，而没有一个残基占主导地位; VH和VL CDR 3结构域都有助于结合的特异性，而CDR 1 L阻碍DNA结合。结果表明静电在DNA与MAb 2C 10的相互作用中起重要作用。(C)1999 Elsevier Science Ltd.保留所有权利。
We have used single and multiple site-directed mutagenesis, and molecular modeling, to identify critical residues in the DNA binding site of MAb 2C10, an IgG anti-dsDNA autoantibody from an MRL/lpr lupus mouse. Simultaneous replacement of four Arg residues in the CDR3H abolished binding activity. With one exception, replacement of any one of these Arg residues reduced the activity to 20-50% of the unmutated scFv activity. Arg to Asp replacements had a slightly greater effect than Arg to Ala replacements. In the one exceptional case, replacement of Arg99 with Ala actually increased DNA binding five-fold and replacement by Asp had little effect. Mutation of Phe32 and Asn35 to Ala in CDR1H decreased DNA binding, whereas replacement of Arg31 with Ala had negligible effect. Ala substitution of any one of a cluster of Asp residues in CDR1L increased DNA binding three to six-fold, confirming previous findings that the L-chain of MAb 2C10 is not favorable for DNA binding. The L-chain does participate in shaping the selectivity of antigen binding, and mutation of CDR3L residue Asp92 or Asn93 caused a decrease in DNA binding activity. Directed mutagenesis, consistent with a molecular model, indicates that: several CDR amino acids contribute to DNA binding, without one residue dominating; both VH and VL CDR3 domains contribute to specificity of binding whereas the CDR1L hinders DNA binding. The results suggest a significant role for electrostatics in the interaction of DNA with MAb 2C10. (C) 1999 Elsevier Science Ltd. All rights reserved.