Swint-Kruse Liskin, Larson Christopher, Pettitt B Montgomery, Matthews Kathleen Shive
Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005, USA.
Protein Sci. 2002 Apr;11(4):778-94. doi: 10.1110/ps.4050102.
LacI and PurR are highly homologous proteins. Their functional units are homodimers, with an N-terminal DNA binding domain that comprises the helix-turn-helix (HTH), N-linker, and hinge regions from both monomers. Hinge structural changes are known to occur upon DNA dissociation but are difficult to monitor experimentally. The initial steps of hinge unfolding were therefore examined using molecular dynamics simulations, utilizing a truncated, chimeric protein comprising the LacI HTH/N-linker and PurR hinge. A terminal Gly-Cys-Gly was added to allow "dimerization" through disulfide bond formation. Simulations indicate that differences in LacI and PurR hinge primary sequence affect the quaternary structure of the hinge x hinge' interface. However, these alternate hinge orientations would be sterically restricted by the core domain. These results prompted detailed comparison of recently available DNA-bound structures for LacI and truncated LacI(1-62) with the PurR structure. Examination revealed that different N-linker and hinge contacts to the core domain of the partner monomer (which binds effector molecule) affect the juxtapositions of the HTH, N-linker, and hinge regions in the DNA binding domain. In addition, the two full-length repressors exhibit significant differences in the interactions between the core and the C-linker connection to the DNA binding domain. Both linkers and the hinge have been implicated in the allosteric response of these repressors. Intriguingly, one functional difference between these two proteins is that they exhibit opposite allosteric response to effector. Simulations and observed structural distinctions are correlated with mutational analysis and sequence information from the LacI/GalR family to formulate a mechanism for fine-tuning individual repressor function.
LacI和PurR是高度同源的蛋白质。它们的功能单元是同型二聚体，其N端DNA结合结构域包含来自两个单体的螺旋-转角-螺旋（HTH）、N-连接区和铰链区。已知铰链结构变化会在DNA解离时发生，但很难通过实验监测。因此，利用分子动力学模拟研究了铰链展开的初始步骤，使用了一种截短的嵌合蛋白，该蛋白包含LacI的HTH/N-连接区和PurR的铰链区。添加了一个末端甘氨酸-半胱氨酸-甘氨酸，以通过二硫键形成实现“二聚化”。模拟表明，LacI和PurR铰链一级序列的差异会影响铰链x铰链'界面的四级结构。然而，这些交替的铰链方向会受到核心结构域的空间限制。这些结果促使对最近获得的LacI和截短的LacI(1-62)与PurR结构的DNA结合结构进行详细比较。研究发现，与结合效应分子的伙伴单体的核心结构域的不同N-连接区和铰链接触会影响DNA结合结构域中HTH、N-连接区和铰链区的并列情况。此外，两种全长阻遏蛋白在核心与连接到DNA结合结构域的C-连接区之间的相互作用上表现出显著差异。连接区和铰链都与这些阻遏蛋白的变构反应有关。有趣的是，这两种蛋白质之间的一个功能差异是它们对效应物表现出相反的变构反应。模拟和观察到的结构差异与LacI/GalR家族的突变分析和序列信息相关联，以制定一种微调单个阻遏蛋白功能的机制。
