Department of Chemistry, University of Toronto, 3359 Mississauga Road, Mississauga, Ontario L5L 1C6, Canada.
Biochemistry. 2012 Aug 21;51(33):6632-43. doi: 10.1021/bi300718f. Epub 2012 Aug 10.
Crystal structures of the GCN4 bZIP (basic region/leucine zipper) with the AP-1 or CRE site show how each GCN4 basic region binds to a 4 bp cognate half-site as a single DNA target; however, this may not always fully describe how bZIP proteins interact with their target sites. Previously, we showed that the GCN4 basic region interacts with all 5 bp in half-site TTGCG (termed 5H-LR) and that 5H-LR comprises two 4 bp subsites, TTGC and TGCG, which individually are also target sites of the basic region. In this work, we explore how the basic region interacts with 5H-LR when the bZIP dimer localizes to full-sites. Using AMBER molecular modeling, we simulated GCN4 bZIP complexes with full-sites containing 5H-LR to investigate in silico the interface between the basic region and 5H-LR. We also performed in vitro investigation of bZIP-DNA interactions at a number of full-sites that contain 5H-LR versus either subsite: we analyzed results from DNase I footprinting and electrophoretic mobility shift assay (EMSA) and from EMSA titrations to quantify binding affinities. Our computational and experimental results together support a highly dynamic DNA-binding model: when a bZIP dimer localizes to its target full-site, the basic region can alternately recognize either subsite as a distinct target at 5H-LR and translocate between the subsites, potentially by sliding and hopping. This model provides added insights into how α-helical DNA-binding domains of transcription factors can localize to their gene regulatory sequences in vivo.
GCN4 bZIP（碱性区/亮氨酸拉链）与 AP-1 或 CRE 位点的晶体结构表明，每个 GCN4 碱性区如何作为单个 DNA 靶标与 4 bp 同源半位点结合；然而，这可能并不完全描述 bZIP 蛋白如何与其靶位点相互作用。以前，我们表明 GCN4 碱性区与半位点 TTGCG（称为 5H-LR）中的所有 5 bp 相互作用，并且 5H-LR 包含两个 4 bp 亚位点 TTGC 和 TGCG，它们各自也是碱性区的靶位点。在这项工作中，我们探讨了当 bZIP 二聚体定位到全长位点时，碱性区如何与 5H-LR 相互作用。我们使用 AMBER 分子建模模拟了包含 5H-LR 的全长位点的 GCN4 bZIP 复合物，以在计算机上研究碱性区和 5H-LR 之间的界面。我们还在包含 5H-LR 的许多全长位点上进行了 bZIP-DNA 相互作用的体外研究，以研究包含亚位点的情况：我们分析了来自 DNase I 足迹法和电泳迁移率变动分析（EMSA）的结果，以及来自 EMSA 滴定的结果，以量化结合亲和力。我们的计算和实验结果共同支持了一种高度动态的 DNA 结合模型：当 bZIP 二聚体定位到其靶全长位点时，碱性区可以交替识别 5H-LR 上的任一亚位点作为不同的靶标，并在亚位点之间易位，可能通过滑动和跳跃。该模型提供了对转录因子的α-螺旋 DNA 结合域如何在体内定位到其基因调节序列的深入了解。
