Department of Environmental Protection, Estación Experimental del Zaidín-Consejo Superior de Investigaciones Científicas, 18008 Granada, Spain.
Proc Natl Acad Sci U S A. 2011 Sep 13;108(37):15372-7. doi: 10.1073/pnas.1018894108. Epub 2011 Aug 29.
Members of the IclR family control bacterial genes involved in a number of physiological processes. The IclR-family member TtgV crystallizes as a tetramer, with each TtgV monomer consisting of two domains--a DNA binding domain and an effector recognition domain, which are interconnected by an extended α-helix. When bound to DNA, a kink is introduced so that the extended helix is split in two α-helices (helix-4 and -5). Differential scanning calorimetry studies revealed that TtgV unfolds in a single event, suggesting that the two domains unfold cooperatively. When mutations are introduced in helix-5 that disrupt interactions between Arg98 and Glu102, the thermal unfolding of the TtgV domains becomes uncoupled without compromising effector binding. Two of these mutants (TtgVE102R and TtgVE102A) showed impaired release from target DNA, suggesting that these mutations alter signal transmission. By combining various mutants, we found that the mutations in the connecting α-helix exhibited a dominant effect over mutations in DNA binding and effector binding domains. We propose a model in which the loss of cooperativity of unfolding of TtgV reflects perturbed interdomain communication, and that the transition from the continuous to discontinuous helix may mediate interdomain communication necessary for the proper functioning of TtgV.
IclR 家族成员控制着参与多种生理过程的细菌基因。TtgV 是 IclR 家族的成员，它以四聚体的形式结晶，每个 TtgV 单体由两个结构域组成——DNA 结合结构域和效应物识别结构域，它们通过一个伸展的α-螺旋相互连接。当与 DNA 结合时，会引入一个扭曲，使伸展的螺旋分裂成两个α-螺旋（螺旋-4 和 -5）。差示扫描量热法研究表明，TtgV 以单一事件展开，表明两个结构域协同展开。当在螺旋-5 中引入破坏 Arg98 和 Glu102 之间相互作用的突变时，TtgV 结构域的热展开会失去偶联而不影响效应物结合。其中两个突变体（TtgVE102R 和 TtgVE102A）显示出从靶 DNA 释放受损，表明这些突变改变了信号传递。通过结合各种突变体，我们发现连接α-螺旋中的突变表现出对 DNA 结合和效应物结合结构域中的突变的显性效应。我们提出了一个模型，其中 TtgV 解折叠的协同性丧失反映了干扰的结构域间通讯，并且从连续到不连续螺旋的转变可能介导了 TtgV 正常功能所需的结构域间通讯。
