Biophysics Group, Department of Physics and Astronomy, Faculty of Sciences, VU University, Amsterdam, The Netherlands.
Biochemistry. 2010 Jun 15;49(23):4752-9. doi: 10.1021/bi100527a.
The bacterium Caulobacter crescentus encodes a two-component signaling protein, LovK, that contains an N-terminal photosensory LOV domain coupled to a C-terminal histidine kinase. LovK binds a flavin cofactor, undergoes a reversible photocycle, and displays regulated ATPase and autophosphorylation activity in response to visible light. Femtosecond to nanosecond visible absorption spectroscopy demonstrates congruence between full-length LovK and isolated LOV domains in the mechanism and kinetics of light-dependent cysteinyl-C4(a) adduct formation and rupture, while steady-state absorption and fluorescence line narrowing (FLN) spectroscopies reveal unique features in the electronic structure of the LovK flavin cofactor. In agreement with other sensor histidine kinases, ATP binds specifically to LovK with micromolar affinity. However, ATP binding to the histidine kinase domain of LovK has no apparent effect on global protein structure as assessed by differential Fourier transform infrared (FTIR) spectroscopy. Cysteinyl adduct formation results in only minor changes in the structure of LovK as determined by differential FTIR. This study provides insight into the structural underpinnings of LOV-mediated signal transduction in the context of a full-length histidine kinase. In particular, the data provide evidence for a model in which small changes in the tertiary/quaternary structure of LovK, as triggered by photon detection in the N-terminal LOV sensory domain, are sufficient to regulate histidine kinase activity.
新月柄杆菌编码一种双组分信号蛋白，LovK，它包含一个 N 端光感受器 LOV 结构域和一个 C 端组氨酸激酶。LovK 结合黄素辅因子，经历可逆的光循环，并对可见光表现出调节的 ATP 酶和自磷酸化活性。飞秒到纳秒的可见吸收光谱证明全长 LovK 和分离的 LOV 结构域在光依赖性半胱氨酸 C4(a)加合物形成和断裂的机制和动力学方面是一致的，而稳态吸收和荧光线窄化（FLN）光谱揭示了 LovK 黄素辅因子电子结构的独特特征。与其他传感器组氨酸激酶一致，ATP 以微摩尔亲和力特异性结合 LovK。然而，ATP 结合到 LovK 的组氨酸激酶结构域对全局蛋白质结构没有明显影响，如差示傅里叶变换红外（FTIR）光谱所评估的那样。半胱氨酸加合物的形成导致 LovK 结构只有微小变化，如差示 FTIR 所确定的那样。这项研究提供了在全长组氨酸激酶背景下 LOV 介导的信号转导的结构基础的深入了解。特别是，数据提供了证据表明，由 N 端 LOV 感觉域中光子检测触发的 LovK 的三级/四级结构的微小变化足以调节组氨酸激酶活性。
