Max-Planck-Institute for Bioinorganic Chemistry, Stifstrasse 34-36 45470, Mülheim, Germany.
Photochem Photobiol Sci. 2010 Jan;9(1):47-56. doi: 10.1039/b9pp00075e. Epub 2009 Nov 30.
YtvA from Bacillus subtilis is a blue-light responsive, flavin-binding photoreceptor, built of a light-sensing LOV domain (aa 25-126) and an NTP (nucleoside triphosphate)-binding STAS domain (aa 147-261). The STAS domain is supposed to be the effector part of the protein or a secondary switch. Both domains are connected by a linker polypeptide. The active form of YtvA is generated upon light excitation, causing the formation of a covalent bond between a cysteine residue (Cys62) in the LOV domain and the position 4a of the flavin chromophore. This photoadduct formation within the LOV domain results in a conformational change of the NTP-binding cavity, evidencing intra-protein signal transmission. We have previously shown that Glu105, localized on the beta-scaffold of the LOV-core, is involved in this process. Here, we extend this work by the identification of further residues that upon mutation supress or strongly impair signal transmission by interfering with the communication between the two domains. These comprise L106 and D109 on the LOV domain; K130 and K134 on the linker region; D193, L194 and G196 within the DLSG GTP-binding motif (switch region) and N201 on the STAS domain. Furthermore in the mutated S195A and D193A proteins, GTP affinity is diminished. Other mutations investigated have little or no effect on signal transmission and GTP-binding affinity: R63K that was found to accelerate the thermal recovery of the parent state ca. ten-fold; K128A, Q129A and Y132A within the linker region, and S183A and S212A on the STAS domain. The results show a key role of the LOV domain beta-scaffold and of positively charged residues within the linker for intra-protein signal transmission. Furthermore they evidence the conformational switch function of a structurally conserved strand-loop-helix region (bearing the DLSG GTP-binding motif and N201) within the STAS domain that constitutes a novel GTP-binding fold.
枯草芽孢杆菌 YtvA 是一种蓝光响应的黄素结合光受体，由一个光感应 LOV 结构域（aa25-126）和一个 NTP（核苷三磷酸）结合 STAS 结构域（aa147-261）组成。STAS 结构域被认为是蛋白质的效应部分或次级开关。这两个结构域由一个连接多肽连接。YtvA 的活性形式是在光激发下产生的，导致 LOV 结构域中的半胱氨酸残基（Cys62）与黄素发色团的 4a 位之间形成共价键。这种 LOV 结构域内的光加合物形成导致 NTP 结合腔的构象变化，证明了蛋白质内信号传递。我们之前已经表明，位于 LOV 核心β支架上的 Glu105 参与了这个过程。在这里，我们通过鉴定进一步的残基来扩展这项工作，这些残基在突变后通过干扰两个结构域之间的通讯来抑制或严重损害信号传递。这些残基包括 LOV 结构域上的 L106 和 D109；连接区上的 K130 和 K134；DLSG GTP 结合基序（开关区）内的 D193、L194 和 G196；以及 STAS 结构域上的 N201。此外，在突变的 S195A 和 D193A 蛋白中，GTP 亲和力降低。其他研究的突变对信号传递和 GTP 结合亲和力影响很小或没有影响：R63K 被发现使亲本状态的热恢复速度加快约十倍；连接区的 K128A、Q129A 和 Y132A，以及 STAS 结构域上的 S183A 和 S212A。结果表明，LOV 结构域β支架和连接区中的正电荷残基对蛋白质内信号传递起着关键作用。此外，它们证明了 STAS 结构域中一个结构保守的链环螺旋区域（带有 DLSG GTP 结合基序和 N201）的构象开关功能，该区域构成了一种新的 GTP 结合折叠。
