Structural basis of Rap phosphatase inhibition by Phr peptides.
Structural basis of Rap phosphatase inhibition by Phr peptides.
复制标题
DOI：
10.1371/journal.pbio.1001511
复制
发表时间：
2013
期刊：
影响因子：
9.8
通讯作者：
Marina A
中科院分区：
文献类型：
--
作者：
Gallego del Sol F;Marina A
结构和功能的研究显示了Phr信号肽抑制Rap蛋白的分子机制，为群体感应中的肽识别和歧视提供了新的见解。双组分系统是细菌中主要的信号转导装置，由组氨酸激酶和效应反应调节因子（RR）组成。在芽孢杆菌中，Rap蛋白家族通过抑制参与这些途径的RR组分来调节由双组分系统介导的复杂信号传导过程，例如感受态、孢子形成或生物膜形成。尽管具有高度的序列同源性，但Rap蛋白通过两种完全不同的作用机制发挥其活性：诱导RR去磷酸化或阻断RR与其靶启动子的结合。然而，涉及Rap蛋白的调节机制甚至更加复杂，因为Rap活性通过在分子水平上仍然未知的机制被特异性信号肽（Phr）拮抗。使用X-射线分析，我们确定了RapF的结构，抗激活剂的能力RR ComA，单独和复杂的调节肽PhrF。本文提供的结构和功能数据揭示肽PhrF通过变构机制阻断RapF-ComA相互作用。PhrF通过诱导RapF的缩窄（constriction）来容纳RapF的C-末端tetratricopeptide重复结构域，所述缩窄是通过显著旋转传播到N-末端ComA结合结构域的构象变化。这种运动部分破坏了ComA的结合位点，触发的ComA解离，其与RapF的相互作用也是空间位阻受损的PhrF诱导的构象的RapF。RapF-PhrF结构指导下的Rap蛋白的序列分析揭示了Phr识别和区分的分子基础，使我们能够通过单个残基的改变来放松RapF的Phr特异性。在微生物中，双组分信号系统被广泛用于感知和响应环境变化，包括Phr寡肽的群体感应。虽然这些系统所需的最低限度的机器包括一个传感器组氨酸激酶和一个效应反应调节器（RR），辅助蛋白，称为“连接器”，能够调节这种机器的活动，正在出现作为额外的球员在这个复杂的信号传导过程。RAP蛋白是原型连接器，能够通过使RR去磷酸化或通过物理阻断它们来调节RR的活性。Rap蛋白本身又被特定的Phr肽抑制，增加了额外的复杂性，但Rap蛋白如何被其同源的Phr肽调节仍然未知。为了回答这个问题，我们解决了RapF的结构，RapF是Rap家族成员，可以单独阻断RR ComA，也可以与其抑制肽PhrF复合。我们的结构和功能的结果表明，PhrF块RapF-ComA相互作用的变构机制，因为PhrF-RapF相互作用诱导的构象变化，传播到的ComA结合位点，破坏它，并触发解离的ComA RapF。使用我们的结构指导的序列分析，我们分别确定了负责肽锚和特异性的残基组，并且能够简单地通过改变单个残基来放松RapF-Phr特异性。这些关键残基和Rap抑制机制的知识开辟了重新设计Rap蛋白的可能性，并为生物和生物技术应用重新编程信号通路铺平了道路。
A structural and functional study shows the molecular mechanism of Rap protein inhibition by Phr signaling peptides, providing new insights into peptide recognition and discrimination in quorum sensing. Two-component systems, composed of a sensor histidine kinase and an effector response regulator (RR), are the main signal transduction devices in bacteria. In Bacillus, the Rap protein family modulates complex signaling processes mediated by two-component systems, such as competence, sporulation, or biofilm formation, by inhibiting the RR components involved in these pathways. Despite the high degree of sequence homology, Rap proteins exert their activity by two completely different mechanisms of action: inducing RR dephosphorylation or blocking RR binding to its target promoter. However the regulatory mechanism involving Rap proteins is even more complex since Rap activity is antagonized by specific signaling peptides (Phr) through a mechanism that remains unknown at the molecular level. Using X-ray analyses, we determined the structure of RapF, the anti-activator of competence RR ComA, alone and in complex with its regulatory peptide PhrF. The structural and functional data presented herein reveal that peptide PhrF blocks the RapF-ComA interaction through an allosteric mechanism. PhrF accommodates in the C-terminal tetratricopeptide repeat domain of RapF by inducing its constriction, a conformational change propagated by a pronounced rotation to the N-terminal ComA-binding domain. This movement partially disrupts the ComA binding site by triggering the ComA disassociation, whose interaction with RapF is also sterically impaired in the PhrF-induced conformation of RapF. Sequence analyses of the Rap proteins, guided by the RapF-PhrF structure, unveil the molecular basis of Phr recognition and discrimination, allowing us to relax the Phr specificity of RapF by a single residue change. In microorganisms, two component signaling systems are widely used to sense and respond to environmental changes, including quorum-sensing of Phr oligopeptides. Although the minimal machinery required for these systems comprises a sensor histidine kinase and an effector response regulator (RR), ancillary proteins, termed “connectors,” capable of modulating the activity of this machinery, are emerging as additional players in this complex signaling process. Rap proteins are archetypal connectors, able to modulate the activity of RRs either by dephosphorylating them or by physically blocking them. Rap proteins are themselves in turn inhibited by specific Phr peptides, adding an extra level of complexity, but how a Rap protein is regulated by its cognate Phr peptide remains unknown. To answer this question, we solved the structure of RapF, a Rap family member that blocks RR ComA, alone and in the complex with its inhibitory peptide PhrF. Our structural and functional results reveal that PhrF blocks the RapF-ComA interaction by an allosteric mechanism since the PhrF-RapF interaction induces a conformational change that is propagated to the the ComA binding site, disrupting it and triggering the dissociation of ComA from RapF. Using sequence analysis guided by our structure, we pinpointed sets of residues responsible for peptide anchor and specificity, respectively, and were able to relax RapF-Phr specificity simply by changing a single residue. Knowledge of these key residues and the Rap inhibition mechanism opens up the possibility of re-engineering Rap proteins, and paves the way to reprogramming signaling pathways for biological and biotechnological applications.
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
DOI：
10.1107/s0907444905001307
发表时间：
2005-04-01
影响因子：
2.2
作者：
Panjikar, S;Parthasarathy, V;Tucker, PA
通讯作者：
Tucker, PA
影响因子：
3.6
作者：
Kozlowicz, Briana K.;Shi, Ke;Dunny, Gary M.
通讯作者：
Dunny, Gary M.
影响因子：
14.9
作者：
Davis IW;Leaver-Fay A;Chen VB;Block JN;Kapral GJ;Wang X;Murray LW;Arendall WB 3rd;Snoeyink J;Richardson JS;Richardson DC
通讯作者：
Richardson DC
DOI：
10.1073/pnas.94.16.8612
发表时间：
1997-08-05
影响因子：
11.1
作者：
Perego, M
通讯作者：
Perego, M