Laity J H, Dyson H J, Wright P E
Department of Molecular Biology, Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037, USA.
J Mol Biol. 2000 Jan 28;295(4):719-27. doi: 10.1006/jmbi.1999.3406.
High-affinity, sequence-specific DNA binding by Cys(2)-His(2) zinc finger proteins is mediated by both specific protein-base interactions and non-specific contacts between charged side-chains and the phosphate backbone. In addition, in DNA complexes of multiple zinc fingers, protein-protein interactions between the finger units contribute to the binding affinity. We present NMR evidence for another contribution to high- affinity binding, a highly specific DNA-induced helix capping involving residues in the linker sequence between fingers. Capping at the C terminus of the alpha-helix in each zinc finger, incorporating a consensus TGEKP linker sequence that follows each finger, provides substantial binding energy to the DNA complexes of zinc fingers 1-3 of TFIIIA (zf1-3) and the four zinc fingers of the Wilms' tumor suppressor protein (wt1-4). The same alpha-helix C-capping motif is observed in the X-ray structures of four other protein-DNA complexes. The structures of each of the TGEKP linkers in these complexes can be superimposed on the linker sequences in the zf1-3 complex, revealing a remarkable similarity in both backbone and side-chain conformations. The canonical linker structures from the zinc-finger-DNA complexes have been compared to the NMR structure of the TGEKP linker connecting fingers 1 and 2 in zf1-3 in the absence of DNA. This comparison reveals that additional stabilization likely arises in the DNA complexes from hydrogen bonding between the backbone amide of E3 and the side-chain O(gamma) of T1 in the linker. We suggest that these DNA-induced C-capping interactions provide a means whereby the multiple-finger complex, which must necessarily be domain-flexible in the unbound state as it searches for the correct DNA sequence, can be "snap-locked" in place once the correct DNA sequence is encountered. These observations provide a rationale for the high conservation of the TGEKP linker sequences in Cys(2)-His(2) zinc finger proteins.
半胱氨酸（2）-组氨酸（2）锌指蛋白与DNA的高亲和力、序列特异性结合是由特定的蛋白质-碱基相互作用以及带电荷的侧链与磷酸骨架之间的非特异性接触介导的。此外，在多个锌指的DNA复合物中，指单元之间的蛋白质-蛋白质相互作用有助于结合亲和力。我们提供了核磁共振证据，证明了对高亲和力结合的另一种贡献，即一种高度特异性的DNA诱导的螺旋封端，涉及手指之间连接序列中的残基。在每个锌指的α-螺旋C末端进行封端，包含每个手指后面的共有TGEKP连接序列，为TFIIIA的锌指1-3（zf1-3）和威尔姆斯肿瘤抑制蛋白的四个锌指（wt1-4）的DNA复合物提供了大量的结合能量。在其他四个蛋白质-DNA复合物的X射线结构中也观察到了相同的α-螺旋C封端基序。这些复合物中每个TGEKP连接子的结构都可以叠加在zf1-3复合物中的连接序列上，揭示了主链和侧链构象的显著相似性。已将锌指-DNA复合物的典型连接子结构与不存在DNA时zf1-3中连接手指1和2的TGEKP连接子的核磁共振结构进行了比较。这种比较表明，DNA复合物中可能通过连接子中E3的主链酰胺与T1的侧链O（γ）之间的氢键产生额外的稳定性。我们认为，这些DNA诱导的C封端相互作用提供了一种方式，通过这种方式，在未结合状态下必然具有结构域灵活性以寻找正确DNA序列的多手指复合物，一旦遇到正确的DNA序列，就可以“快速锁定”到位。这些观察结果为半胱氨酸（2）-组氨酸（2）锌指蛋白中TGEKP连接序列的高度保守性提供了理论依据。
