Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3.
Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3.
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DOI：
10.1371/journal.pgen.1000352
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发表时间：
2009-01
期刊：
影响因子：
4.5
通讯作者：
Quail, Peter H.
中科院分区：
文献类型：
--
作者：
Kikis, Elise A.;Oka, Yoshito;Hudson, Matthew E.;Nagatani, Akira;Quail, Peter H.
BHLH转录因子光敏色素相互作用因子3(PIF3)与光激活的PfR形式的拟南芥光敏色素B(PhyB)特异地相互作用。这种相互作用在体内诱导PIF3的磷酸化和降解，并调节PhyB介导的幼苗对红光的脱黄化反应。为了确定在PhyB N末端区域中破坏这种相互作用的错义突变，我们开发了一种酵母反向杂交筛选。在体外免疫共沉淀实验中，在此筛选中或在先前的拟南芥突变体的遗传筛选中发现的15个单独的突变对红光的敏感性降低，也被证明也破坏了光诱导的PhyB与PIF3的结合。这些PhyB错义突变体一般分为两类：I类(11个突变体)包含由于生色团连接或光转化而导致的光信号感知缺陷；II类(4个突变体)包含信号感知正常但将信号转导到PIF3的能力缺陷的突变体。通过建立拟南芥PhyB发色团结合区三维结构的同源模型，基于耐辐射奇球菌光敏色素的晶体结构，我们预测四个II类突变的PhyB残基中有三个是在PAS和GAF结构域之间的裂隙中暴露的溶剂。这一推论表明，PhyB与PIF3的物理相互作用可能直接需要这些残基。由于这三个残基也是PhyB抑制下胚轴伸长对红光的反应所必需的，它们在功能上是从光激活的PhyB向PIF3和其他相关的bHLH转录因子传递信号所必需的，也是参与调节幼苗脱黄化的其他下游信号成分所必需的。植物监测其环境中的信息光信号，这些光信号用于指导适应性形态发生反应。光敏色素(PHY)光感受器家族在这一过程中起着中心作用。光感受后，PHY分子迅速移位到细胞核，在那里它们与被称为PIF(PHY相互作用因子)的基本螺旋-环-螺旋转录因子相互作用，并诱导控制形态发生反应的基因表达变化。PHY蛋白中负责从激活的光感受器直接向转导伙伴传递分子间信号的分子决定因素尚不确定。利用拟南芥PhyB的随机突变，结合反向杂交蛋白相互作用筛选，我们鉴定了N-末端区域的错义突变，这些突变取消了光感受器分子与PIF3的结合。这些突变的PhyB分子的一个子集保留了感知光信号的能力，但在将该信号转导到PIF3和其他相关PIF的能力上存在缺陷。据预测，这些分子中的突变残基将聚集在蛋白质的表面，形成一种被称为“光感应结”的结构。这些残基对于在活的植物中由PhyB调控的生长是必要的，从而确定所确定的蛋白质区域似乎作为分子界面的一个组成部分，负责将信号直接传递到细胞中的转导伙伴。
The bHLH transcription factor, PHYTOCHROME INTERACTING FACTOR 3 (PIF3), interacts specifically with the photoactivated, Pfr, form of Arabidopsis phytochrome B (phyB). This interaction induces PIF3 phosphorylation and degradation in vivo and modulates phyB-mediated seedling deetiolation in response to red light. To identify missense mutations in the phyB N-terminal domain that disrupt this interaction, we developed a yeast reverse-hybrid screen. Fifteen individual mutations identified in this screen, or in previous genetic screens for Arabidopsis mutants showing reduced sensitivity to red light, were shown to also disrupt light-induced binding of phyB to PIF3 in in vitro co-immunoprecipitation assays. These phyB missense mutants fall into two general classes: Class I (eleven mutants) containing those defective in light signal perception, due to aberrant chromophore attachment or photoconversion, and Class II (four mutants) containing those normal in signal perception, but defective in the capacity to transduce this signal to PIF3. By generating a homology model for the three-dimensional structure of the Arabidopsis phyB chromophore-binding region, based on the crystal structure of Deinococcus radiodurans phytochrome, we predict that three of the four Class II mutated phyB residues are solvent exposed in a cleft between the presumptive PAS and GAF domains. This deduction suggests that these residues could be directly required for the physical interaction of phyB with PIF3. Because these three residues are also necessary for phyB-imposed inhibition of hypocotyl elongation in response to red light, they are functionally necessary for signal transfer from photoactivated phyB, not only to PIF3 and other related bHLH transcription factors tested here, but also to other downstream signaling components involved in regulating seedling deetiolation. Plants monitor their environment for informational light signals that are used to direct adaptive morphogenic responses. The phytochrome (phy) family of photoreceptors are central to this process. Upon photoperception, phy molecules rapidly translocate to the nucleus where they interact with basic helix-loop-helix transcription factors, termed PIFs (phy-Interacting Factors), and induce gene-expression changes that control morphogenic responses. The molecular determinants in the phy protein responsible for direct intermolecular signal transfer from the activated photoreceptor to transduction partners are undefined. Using random mutagenesis of Arabidopsis phyB, coupled with a reverse-hybrid protein-interaction screen, we identified missense mutations in the N-terminal domain that abrogate the binding of the photoreceptor molecule to PIF3. A subset of these mutated phyB molecules retain the capacity for light-signal perception but are defective in the capacity to transduce that signal to PIF3 and other related PIFs. The mutated residues in these molecules are predicted to cluster at the surface of the protein in a structure termed the “light-sensing knot.” These residues are necessary for phyB-regulated growth in the living plant, establishing that the protein region identified appears to function as a component of the molecular interface responsible for direct signal transfer to transduction partners in the cell.
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影响因子：
9.2
作者：
Leivar, Pablo;Monte, Elena;Oka, Yoshito;Liu, Tiffany;Carle, Christine;Castillon, Alicia;Huq, Enamul;Quail, Peter H.
通讯作者：
Quail, Peter H.
DOI：
10.1073/pnas.1935989100
发表时间：
2003-11-25
影响因子：
11.1
作者：
Chen, M;Schwabb, R;Chory, J
通讯作者：
Chory, J
影响因子：
11.6
作者：
Khanna, Rajnish;Shen, Yu;Quail, Peter H.
通讯作者：
Quail, Peter H.
DOI：
10.1073/pnas.140520097
发表时间：
2000-07-05
影响因子：
11.1
作者：
Krall, L;Reed, JW
通讯作者：
Reed, JW
影响因子：
14.9
作者：
Leanna, CA;Hannink, M
通讯作者：
Hannink, M