Functional consequences of exchanging domains between LacI and PurR are mediated by the intervening linker sequence.
Functional consequences of exchanging domains between LacI and PurR are mediated by the intervening linker sequence.
复制标题
LacI 和 PurR 之间交换结构域的功能后果是由插入的接头序列介导的。
DOI：
10.1002/prot.21412
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发表时间：
2007
期刊：
影响因子：
2.9
通讯作者：
Swint-Kruse,Liskin
中科院分区：
文献类型：
--
作者：
Tungtur,Sudheer;Egan,SusanM;Swint-Kruse,Liskin
同源功能可以通过改变影响结合位点或长程相互作用的残基来区分。LacI和PurR是代表细菌转录调节因子的LacI/GalR家族（>500个成员）的两种蛋白质。所有成员都有不同的DNA结合和调节结构域，由大约18个氨基酸连接。每个同源物对不同的DNA和调节效应配体具有特异性; LacI和PurR在DNA和效应结合位点之间的变构通信中也表现出差异。LacI和PurR的比较研究表明，调节结构域和接头之间的界面的改变是重要的区分其功能。四个残基（相当于LacI位置48，55，58和61）似乎特别重要的创造一个独特的接口，并预测是必要的变构调节。然而，接头中的邻近残基与DNA配体相互作用。因此，观察到的接头和调节结构域之间的相互作用的差异可能是改变功能的原因或两种蛋白质结合不同的DNA配体的影响。为了分离这些可能性，我们创建了具有LacI DNA结合结构域/接头和PurR调节结构域（LLhP）的嵌合蛋白。如果界面需要同源物特异性相互作用以传播来自效应物结合的信号，则LLhP抑制不应受效应物结合的变构调节。实验表明，LLhP能够从lacO 1阻遏，与预期相反，变构反应是完整的。此外，通过LLhP接头中的取代恢复PurR样相互作用的可能性倾向于减少抑制。这些影响对于残基58和61尤其明显。很明显，LLhP对lacO 1DNA位点的结合亲和力对接头的长距离变化很敏感。这一结果也提出了58和61位突变与DNA结合位点变化共同进化的可能性。此外，在不存在和存在效应配体的情况下测量的抑制显示，对于在位置48和55处具有取代的几种LLhP变体，变构反应增加。因此，虽然这些位点的侧链变异通常不决定变构的存在或不存在，但氨基酸的性质可以调节对效应物的应答。Proteins 2007.© 2007 Wiley利斯公司
Homologue function can be differentiated by changing residues that affect binding sites or long‐range interactions. LacI and PurR are two proteins that represent the LacI/GalR family (>500 members) of bacterial transcription regulators. All members have distinct DNA‐binding and regulatory domains linked by ∼18 amino acids. Each homologue has specificity for different DNA and regulatory effector ligands; LacI and PurR also exhibit differences in allosteric communication between DNA and effector binding sites. A comparative study of LacI and PurR suggested that alterations in the interface between the regulatory domain and linker are important for differentiating their functions. Four residues (equivalent to LacI positions 48, 55, 58, and 61) appear particularly important for creating a unique interface and were predicted to be necessary for allosteric regulation. However, nearby residues in the linker interact with DNA ligand. Thus, differences observed in interactions between linker and regulatory domain may be thecauseof altered function or aneffectof the two proteins binding different DNA ligands. To separate these possibilities, we created a chimeric protein with the LacI DNA‐binding domain/linker and the PurR regulatory domain (LLhP). If the interface requires homologue‐specific interactions in order to propagate the signal from effector binding, then LLhP repression should not be allosterically regulated by effector binding. Experiments show that LLhP is capable of repression fromlacO1and, contrary to expectation, allosteric response is intact. Further, restoring the potential for PurR‐like interactions via substitutions in the LLhP linker tends todiminishrepression. These effects are especially pronounced for residues 58 and 61. Clearly, binding affinity of LLhP for thelacO1DNA site is sensitive to long‐range changes in the linker. This result also raises the possibility that mutations at positions 58 and 61 co‐evolved with changes in the DNA‐binding site. In addition, repression measured in the absence and presence of effector ligand shows that allosteric response increases for several LLhP variants with substitutions at positions 48 and 55. Thus, while side chain variation at these sites does not generally dictate the presence or absence of allostery, the nature of the amino acid can modulate the response to effector. Proteins 2007. © 2007 Wiley‐Liss, Inc.