Malachite green mediates homodimerization of antibody VL domains to form a fluorescent ternary complex with singular symmetric interfaces.
Malachite green mediates homodimerization of antibody VL domains to form a fluorescent ternary complex with singular symmetric interfaces.
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DOI：
10.1016/j.jmb.2013.08.014
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发表时间：
2013-11-15
影响因子：
5.6
通讯作者：
Bruchez, Marcel P.
中科院分区：
文献类型：
--
作者：
Szent-Gyorgyi, Chris;Stanfield, Robyn L.;Andreko, Susan;Dempsey, Alison;Ahmed, Mushtaq;Capek, Sarah;Waggoner, Alan S.;Wilson, Ian A.;Bruchez, Marcel P.
关键词：
我们报道了一种对称的小分子配体介导抗体轻链可变区（VL）组装成具有新颖界面的相应对称三元复合物。L5* 荧光激活蛋白（FAP）是结合孔雀石绿色染料（MG）以激活强荧光的VL结构域。配体L5* 的晶体学揭示了具有包容性C2对称性的2：1蛋白质：配体复合物，其中MG几乎完全包封在两个VL结构域的反平行排列之间。未配体的L5* VL结构域结晶为类似的反平行VL/VL同二聚体。互补决定区（CDR）在空间上定向以形成新的VL/VL和VL/配体界面，其紧密地约束MG的螺旋桨构象异构体。结合平衡分析表明，高度合作的组装，形成一个非常稳定的VL/MG/VL复合物，使MG的行为作为一个强大的化学诱导剂的二聚化。将两个VL结构域融合到单个蛋白质中使MG结合收紧超过1，000倍至低皮摩尔亲和力，而不改变大的结合焓，表明与配体的键合相互作用和结构域移动的限制对结合做出独立贡献。对称荧光团的荧光激活提供了用于三元复合物的分离和定向进化的选择机制，其中非天然对称结合界面优于典型抗体界面。如L5* 所例示的，这些自报告复合物可用作蛋白质缔合的调节剂或用作高亲和力蛋白质标签和捕获试剂。
We report that a symmetric small molecule ligand mediates the assembly of antibody light chain variable domains (VLs) into a correspondent symmetric ternary complex with novel interfaces. The L5* Fluorogen Activating Protein (FAP) is a VL domain that binds malachite green dye (MG) to activate intense fluorescence. Crystallography of liganded L5* reveals a 2:1 protein:ligand complex with inclusive C2 symmetry, where MG is almost entirely encapsulated between an antiparallel arrangement of the two VL domains. Unliganded L5* VL domains crystallize as a similar antiparallel VL/VL homodimer. The complementarity determining regions (CDRs) are spatially oriented to form novel VL/VL and VL/ligand interfaces that tightly constrain a propeller conformer of MG. Binding equilibrium analysis suggests highly cooperative assembly to form a very stable VL/MG/VL complex, such that MG behaves as a strong chemical inducer of dimerization. Fusion of two VL domains into a single protein tightens MG binding over 1,000-fold to low picomolar affinity without altering the large binding enthalpy, suggesting that bonding interactions with ligand and restriction of domain movements make independent contributions to binding. Fluorescence activation of a symmetrical fluorogen provides a selection mechanism for the isolation and directed evolution of ternary complexes where unnatural symmetric binding interfaces are favored over canonical antibody interfaces. As exemplified by L5*, these self-reporting complexes may be useful as modulators of protein association or as high affinity protein tags and capture reagents.
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DOI：
10.1111/tra.12019
发表时间：
2013-01
期刊：
Traffic (Copenhagen, Denmark)
影响因子：
--
作者：
Bachert C;Linstedt AD
通讯作者：
Linstedt AD
影响因子：
4.7
作者：
Fitzpatrick JA;Yan Q;Sieber JJ;Dyba M;Schwarz U;Szent-Gyorgyi C;Woolford CA;Berget PB;Waggoner AS;Bruchez MP
通讯作者：
Bruchez MP
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
5.6
作者：
Abhinandan, K. R.;Martin, Andrew C. R.
通讯作者：
Martin, Andrew C. R.
影响因子：
5.6
作者：
Dey, Sucharita;Pal, Arumay;Janin, Joel
通讯作者：
Janin, Joel