The Kinetic and Molecular Basis for the Interaction of LexA and Activated RecA Revealed by a Fluorescent Amino Acid Probe.,ACS Chemical Biology

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The Kinetic and Molecular Basis for the Interaction of LexA and Activated RecA Revealed by a Fluorescent Amino Acid Probe.
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2020-02-05 , DOI: 10.1021/acschembio.9b00886
Zachary M Hostetler ₁ , Michael B Cory ₂ , Chloe M Jones ₂ , E James Petersson _{3,

4} , Rahul M Kohli _{4,

5}

Affiliation

The bacterial DNA damage response (the SOS response) is a key pathway involved in antibiotic evasion and a promising target for combating acquired antibiotic resistance. Activation of the SOS response is controlled by two proteins: the repressor LexA and the DNA damage sensor RecA. Following DNA damage, direct interaction between RecA and LexA leads to derepression of the SOS response. However, the exact molecular details of this interaction remain unknown. Here, we employ the fluorescent unnatural amino acid acridonylalanine (Acd) as a minimally perturbing probe of the E. coli RecA:LexA complex. Using LexA labeled with Acd, we report the first kinetic model for the reversible binding of LexA to activated RecA. We also characterize the effects that specific amino acid truncations or substitutions in LexA have on RecA:LexA binding strength and demonstrate that a mobile loop encoding LexA residues 75-84 comprises a key recognition interface for RecA. Beyond insights into SOS activation, our approach also further establishes Acd as a sensitive fluorescent probe for investigating the dynamics of protein-protein interactions in other complex systems.

中文翻译：

荧光氨基酸探针揭示了 LexA 和激活的 RecA 相互作用的动力学和分子基础。

细菌 DNA 损伤反应（SOS 反应）是参与抗生素逃逸的关键途径，也是对抗获得性抗生素耐药性的有希望的目标。SOS 反应的激活由两种蛋白质控制：阻遏物 LexA 和 DNA 损伤感受器 RecA。DNA 损伤后，RecA 和 LexA 之间的直接相互作用导致 SOS 反应的去抑制。然而，这种相互作用的确切分子细节仍然未知。在这里，我们使用荧光非天然氨基酸吖啶丙氨酸 (Acd) 作为大肠杆菌 RecA:LexA 复合物的微扰探针。使用标有 Acd 的 LexA，我们报告了 LexA 与激活的 RecA 可逆结合的第一个动力学模型。我们还描述了 LexA 中特定氨基酸截断或替换对 RecA 的影响：LexA 结合强度并证明编码 LexA 残基 75-84 的移动环包含 RecA 的关键识别界面。除了深入了解 SOS 激活之外，我们的方法还进一步确立了 Acd 作为一种灵敏的荧光探针，用于研究其他复杂系统中蛋白质-蛋白质相互作用的动力学。

更新日期：2020-01-30

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