Engineered disulfide linking the hinge regions within lactose repressor dimer increases operator affinity, decreases sequence selectivity, and alters allostery
Engineered disulfide linking the hinge regions within lactose repressor dimer increases operator affinity, decreases sequence selectivity, and alters allostery
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DOI：
10.1021/bi0114067
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发表时间：
2001-12-25
期刊：
影响因子：
2.9
通讯作者：
Matthews, KS
中科院分区：
文献类型：
--
作者：
Falcon, CM;Matthews, KS
该铰链域包含乳糖抑制因子(LacI)的51-60个氨基酸，在其与操纵子DNA的调控相互作用中发挥着重要作用。这一片段使铰链-DNA和铰链-铰链的接触对DNA结合至关重要。此外，这一小区域在将变构反应传递给诱导剂时起着中心作用。通过突变V52C在二聚体内的伙伴铰链之间引入二硫键导致与野生型LacI相比对01操作符DNA的亲和力增加，并取消对诱导剂的变构反应[Falcon，C.M.，Swint-Kruse，L.，和Matthews，K.S.(1997)J.Biol]。化学。272,26818]。我们已经确定，即使当操纵区内的对称性和半位点间距改变时，二硫键连接蛋白的高亲和力也保持不变，而还原蛋白的结合，如野生型LacI，因这些改变而严重减弱。有趣的是，对V52C氧化的诱导剂的变构反应对于一小群操作员变体保持不变。温度研究表明，二硫键的存在改变了蛋白质-DNA相互作用的热力学，与野生型LaCI相比，DeltaC(P)的幅度要小得多。本文的结果表明，铰链区不仅是LacI高亲和力操纵子结合的重要元件，而且是配体结合域之间必要的通讯元件。此外，结果证实DNA序列/构象可以深刻地影响这一原型调节蛋白的变构。
The hinge domain encompasses amino acids 51-60 of lactose repressor (LacI) and plays an important role in its regulatory interaction with operator DNA. This segment makes both hinge-DNA and hinge-hinge' contacts that are critical to DNA binding. Furthermore, this small region serves as a central element in communicating the allosteric response to inducer. Introducing a disulfide bond between partner hinges within a dimer via the mutation V52C results in a protein that has increased affinity for 01 operator DNA compared to wild-type LacI and abolishes allosteric response to inducer [Falcon, C. M., Swint-Kruse, L., and Matthews, K. S. (1997) J. Biol. Chem. 272, 26818]. We have established that this high affinity is maintained for the disulfide-linked protein even when symmetry and half-site spacing within the operator region are altered, whereas binding by the reduced protein, as for wild-type LacI, is severely diminished by these alterations. Interestingly, the allosteric response to inducer for V52C-oxidized remains intact for a small group of operator variants. Temperature studies demonstrate that the presence of the disulfide alters the thermodynamics of the protein-DNA interaction, with a DeltaC(p) of significantly smaller magnitude compared to wild-type LacI. The results presented here establish the hinge region as an important element not only for LacI high-affinity operator binding but also for the essential communication between ligand binding domains. Moreover, the results confirm that DNA sequence/ conformation can profoundly influence allostery for this prototypic regulatory protein.