Structural basis of response regulator inhibition by a bacterial anti-activator protein.
Structural basis of response regulator inhibition by a bacterial anti-activator protein.
复制标题
DOI：
10.1371/journal.pbio.1001226
复制
发表时间：
2011-12
期刊：
影响因子：
9.8
通讯作者：
Neiditch MB
中科院分区：
文献类型：
--
作者：
Baker MD;Neiditch MB
结构-功能研究表明，Rap蛋白具有不同的，不重叠的表面，与不同的细胞靶点相互作用，并且对于抗激活因子RapF，一个表面模拟DNA结合反应调节因子DNA结合结构域，从而在空间上阻止这种转录反式激活因子的活性。反应调节剂ComA，抗激活剂RapF和信号肽PhrF之间的复杂相互作用控制枯草芽孢杆菌中的感受态发育。更具体地说，ComA驱动遗传感受态基因的表达，而RapF抑制ComA与其靶启动子的相互作用。信号肽PhrF在高细胞密度下积累，并通过拮抗RapF和ComA的相互作用上调遗传能力。RapF如何机制性地发挥作用以抑制ComA活性以及PhrF如何反过来拮抗RapF-ComA相互作用尚不清楚。在这里，我们提出的X-射线晶体结构的RapF在复杂的ComA DNA结合域。沿着生物化学和遗传学研究，X射线晶体结构揭示了RapF如何机械地调节ComA功能。有趣的是，我们发现RapF表面模拟DNA来阻断ComA与其靶启动子的结合。此外，RapF是单独或与PhrF复合的单体，并且其在与PhrF结合时经历构象变化，这可能导致ComA从RapF-ComA复合物中解离。最后，我们比较了RapF与ComA DNA结合结构域复合的结构和RapH与Spo0F复合的结构。这种比较表明，RapF和RapH具有惊人相似的整体结构，并且它们已经进化出不同的、不重叠的表面，以与不同的细胞靶点相互作用。据我们所知，这里提供的数据揭示了第一个原子水平的洞察抑制反应调节DNA结合的抗激活剂。影响Rap和Rap样蛋白与其靶结构域相互作用的化合物可用于调节许多芽孢杆菌属物种中的医学和商业上重要的表型，例如B中的孢子形成。炭疽菌和孢子形成以及B产生Cry蛋白内毒素。苏云金杆菌磷酸化后，称为反应调节剂的细菌蛋白质与DNA启动子结合并激活或抑制转录。这些反应调节剂本身由抗激活蛋白调节，抗激活蛋白可以控制反应调节剂活性而不改变其磷酸化状态。我们已经确定了与反应调节剂ComA的DNA结合结构域复合的抗激活剂RapF的X-射线晶体结构。我们的结构-功能研究表明，RapF使用双管齐下的机制破坏了ComA与DNA的结合。首先，RapF表面模拟DNA，并且这种DNA样表面结合几乎所有的ComA DNA结合残基，从而阻断ComA与DNA的相互作用。第二，RapF抑制ComA二聚化。RapF也受到PhrF肽的调节;我们发现RapF-ComA相互作用表面远离拟议的PhrF结合位点。此外，我们发现RapF在与PhrF结合时发生构象变化，这可能导致其从ComA解离。从这些观察结果，我们得出结论，PhrF结合RapF变构触发其从ComA解离。最后，我们比较了RapF/ComA DNA结合域复合物的结构与另一种反应调节剂Spo0F的结构，Spo0F与磷酸酶RapH复合。这表明，虽然RapF和RapH在结构上相似，但它们已经进化出不同的、不重叠的表面，以与它们不同的细胞靶点相互作用。
Structure-function studies reveal that Rap proteins have distinct, nonoverlapping surfaces that interact with different cellular targets, and that for antiactivator RapF, one surface mimics DNA to bind a response regulator DNA binding domain, thereby sterically preventing the activity of this transcription transactivator. The complex interplay between the response regulator ComA, the anti-activator RapF, and the signaling peptide PhrF controls competence development in Bacillus subtilis. More specifically, ComA drives the expression of genetic competence genes, while RapF inhibits the interaction of ComA with its target promoters. The signaling peptide PhrF accumulates at high cell density and upregulates genetic competence by antagonizing the interaction of RapF and ComA. How RapF functions mechanistically to inhibit ComA activity and how PhrF in turn antagonizes the RapF-ComA interaction were unknown. Here we present the X-ray crystal structure of RapF in complex with the ComA DNA binding domain. Along with biochemical and genetic studies, the X-ray crystal structure reveals how RapF mechanistically regulates ComA function. Interestingly, we found that a RapF surface mimics DNA to block ComA binding to its target promoters. Furthermore, RapF is a monomer either alone or in complex with PhrF, and it undergoes a conformational change upon binding to PhrF, which likely causes the dissociation of ComA from the RapF-ComA complex. Finally, we compare the structure of RapF complexed with the ComA DNA binding domain and the structure of RapH complexed with Spo0F. This comparison reveals that RapF and RapH have strikingly similar overall structures, and that they have evolved different, non-overlapping surfaces to interact with diverse cellular targets. To our knowledge, the data presented here reveal the first atomic level insight into the inhibition of response regulator DNA binding by an anti-activator. Compounds that affect the interaction of Rap and Rap-like proteins with their target domains could serve to regulate medically and commercially important phenotypes in numerous Bacillus species, such as sporulation in B. anthracis and sporulation and the production of Cry protein endotoxin in B. thuringiensis. Upon phosphorylation, bacterial proteins called response regulators bind to DNA promoters and activate or repress transcription. These response regulators are themselves regulated by anti-activator proteins, which can control response regulator activity without altering their phosphorylation state. We have determined the X-ray crystal structure of the anti-activator RapF complexed with the DNA-binding domain of the response regulator ComA. Our structure-function studies show that RapF disrupts the binding of ComA to DNA using a two-pronged mechanism. First, a RapF surface mimics DNA, and this DNA-like surface binds to nearly all of the ComA DNA-binding residues, thus blocking ComA's interaction with DNA. Second, RapF inhibits ComA dimerization. RapF is also regulated by the PhrF peptide; we find that the RapF-ComA interaction surface is distant from the proposed PhrF binding site. Furthermore, we found that RapF undergoes a conformational change upon binding to PhrF, which likely causes its dissociation from ComA. From these observations, we conclude that PhrF binding to RapF allosterically triggers its dissociation from ComA. Finally, we compared the RapF/ComA DNA-binding domain complex structure with the structure of another response regulator, Spo0F, complexed with the phosphatase RapH. This reveals that while RapF and RapH are structurally similar, they have evolved distinct, non-overlapping surfaces to interact with their different cellular targets.
登录
查看更多内容
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
3.6
作者：
Chai, Y;Zhu, J;Winans, SC
通讯作者：
Winans, SC
DOI：
10.1099/00221287-136-5-905
发表时间：
1990-05-01
期刊：
JOURNAL OF GENERAL MICROBIOLOGY
影响因子：
--
作者：
GLEAVE, AP;MOUNTAIN, A;THOMAS, CM
通讯作者：
THOMAS, CM
DOI：
10.1107/s0907444909052925
发表时间：
2010-02
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Adams PD;Afonine PV;Bunkóczi G;Chen VB;Davis IW;Echols N;Headd JJ;Hung LW;Kapral GJ;Grosse-Kunstleve RW;McCoy AJ;Moriarty NW;Oeffner R;Read RJ;Richardson DC;Richardson JS;Terwilliger TC;Zwart PH
通讯作者：
Zwart PH
影响因子：
3.2
作者：
ALBANO, M;HAHN, J;DUBNAU, D
通讯作者：
DUBNAU, D