Phosphorylation-independent regulation of the diguanylate cyclase WspR.
Phosphorylation-independent regulation of the diguanylate cyclase WspR.
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DOI：
10.1371/journal.pbio.0060067
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发表时间：
2008-03-25
期刊：
影响因子：
9.8
通讯作者：
Sondermann H
中科院分区：
文献类型：
--
作者：
De N;Pirruccello M;Krasteva PV;Bae N;Raghavan RV;Sondermann H
触发细菌发病机制和生物膜形成的环境信号由环二聚鸟苷单磷酸（c-di-GMP）水平的变化介导，这是一种独特的真细菌第二信使。细胞c-di-GMP浓度的严格调节由二鸟苷酸环化酶和磷酸二酯酶控制，它们分别负责其产生和降解。在这里，我们提出了双鸟苷酸环化酶WspR的晶体结构，一个保守的GGDEF结构域的革兰氏阴性菌的反应调节剂，结合到c-di-GMP在抑制位点。生化分析表明，反馈调节涉及至少三个不同的寡聚状态的形成。通过四聚体组装从活性二聚体转换为产物抑制二聚体，WspR利用一种新的机制通过寡聚化调节其活性。此外，我们的数据表明，这些酶可以被磷酸二酯酶激活。因此，除了通过调节结构域的磷酸化的典型途径之外，产物和酶浓度都有助于c-di-GMP信号传导的协调。结构比较揭示了相似的寡聚状态的GAF结构域，广泛使用的监管结构域的信号分子保守的古细菌到哺乳动物的组件，这表明了类似的调节机制。细菌可以通过表面的定殖和细胞外基质的分泌从单细胞、自由漂浮的行为模式转变为群落生活形式。这个过程称为生物膜形成，被归因于大多数慢性感染，包括肺部，如囊性纤维化患者中发生的。最近，一个小的细胞内信号分子，核苷酸环二聚鸟苷一磷酸（c-di-GMP），和酶的生产和降解已被发现，中继环境线索的变化，分泌，细胞粘附，并最终，生物膜的形成和毒力。我们已经研究了WspR的分子机制和调节模式，WspR是一种来自假单胞菌和相关致病菌的酶，负责产生c-di-GMP和生物膜形成。在其晶体结构和功能测定的基础上，我们阐明了WspR中由c-di-GMP介导的反馈抑制控制的复杂调节机制。我们假设，WspR是引发的（再）激活酶降解的抑制性核苷酸。此外，我们在囊性纤维化患者中经常发现的细菌子集中确定了WspR抑制位点的突变，这表明由修饰的WspR介导的改变的c-di-GMP信号传导可能有助于这些菌株的致病性。此外，我们提出了与GAF结构域，这是广泛使用的保守的调节信号结构域的结构比较，表明了类似的调节机制。我们提出了一个保守的二鸟苷酸环化酶从假单胞菌，负责环二GMP生产和生物膜形成的调节模型，提供了深入了解的分子机制控制细胞信号和毒力。
Environmental signals that trigger bacterial pathogenesis and biofilm formation are mediated by changes in the level of cyclic dimeric guanosine monophosphate (c-di-GMP), a unique eubacterial second messenger. Tight regulation of cellular c-di-GMP concentration is governed by diguanylate cyclases and phosphodiesterases, which are responsible for its production and degradation, respectively. Here, we present the crystal structure of the diguanylate cyclase WspR, a conserved GGDEF domain-containing response regulator in Gram-negative bacteria, bound to c-di-GMP at an inhibitory site. Biochemical analyses revealed that feedback regulation involves the formation of at least three distinct oligomeric states. By switching from an active to a product-inhibited dimer via a tetrameric assembly, WspR utilizes a novel mechanism for modulation of its activity through oligomerization. Moreover, our data suggest that these enzymes can be activated by phosphodiesterases. Thus, in addition to the canonical pathways via phosphorylation of the regulatory domains, both product and enzyme concentration contribute to the coordination of c-di-GMP signaling. A structural comparison reveals resemblance of the oligomeric states to assemblies of GAF domains, widely used regulatory domains in signaling molecules conserved from archaea to mammals, suggesting a similar mechanism of regulation. Bacteria can switch from a single-cell, free-floating behavioral mode to a community life-form via colonization of surfaces and the secretion of an extracellular matrix. This process, called biofilm formation, has been attributed to a majority of chronic infections, including the lungs, as occurs in patients with cystic fibrosis. Recently, a small intracellular signaling molecule, the nucleotide cyclic dimeric guanosine monophosphate (c-di-GMP), and enzymes for its production and degradation have been discovered that relay environmental cues to changes in secretion, cell adhesion and ultimately, biofilm formation and virulence. We have studied the molecular mechanism and mode of regulation of WspR, an enzyme from Pseudomonas and related pathogenic bacteria responsible for the generation of c-di-GMP and biofilm formation. On the basis of its crystal structure and functional assays, we elucidated a sophisticated regulatory mechanism in WspR that is controlled by feedback inhibition mediated by c-di-GMP. We hypothesize that WspR is primed for the (re)activation by enzymatic degradation of the inhibitory nucleotide. In addition, we identified mutations at the inhibitory site of WspR in a subset of bacteria that are frequently found in cystic fibrosis patients, suggesting that altered c-di-GMP signaling, mediated by modified WspR, may contribute to the pathogenicity of these strains. Furthermore, we present a structural comparison with GAF domains, which are widely used conserved regulatory signaling domains, suggesting a similar mechanism of regulation. We present a model for the regulation of a conserved diguanyate cyclase fromPseudomonas that is responsible for cyclic di-GMP production and biofilm formation, providing insight into the molecular mechani7sm controlling cell signaling and virulence.
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影响因子：
3.3
作者：
Goymer, Patrick;Kahn, Sophie G.;Rainey, Paul B.
通讯作者：
Rainey, Paul B.
DOI：
10.1107/s0907444998003254
发表时间：
1998-09-01
期刊：
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY
影响因子：
--
作者：
Brunger, AT;Adams, PD;Warren, GL
通讯作者：
Warren, GL
DOI：
10.1073/pnas.0511090103
发表时间：
2006-02-21
影响因子：
11.1
作者：
Kulesekara, H;Lee, V;Lory, S
通讯作者：
Lory, S
DOI：
10.1107/s0907444902016657
发表时间：
2002-11-01
影响因子：
2.2
作者：
Adams, PD;Grosse-Kunstleve, RW;Terwilliger, TC
通讯作者：
Terwilliger, TC
影响因子：
3.6
作者：
Kirillina, O;Fetherston, JD;Perry, RD
通讯作者：
Perry, RD