The structure of an unconventional HD-GYP protein from Bdellovibrio reveals the roles of conserved residues in this class of cyclic-di-GMP phosphodiesterases.
The structure of an unconventional HD-GYP protein from Bdellovibrio reveals the roles of conserved residues in this class of cyclic-di-GMP phosphodiesterases.
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作者：
Lovering AL;Capeness MJ;Lambert C;Hobley L;Sockett RE
Cyclic-di-GMP是一种几乎无处不在的细菌第二信使，在控制各种过程期间的局部信号传递中非常重要，包括毒力和基于生物膜的生活方式之间的转换。环状-二-GMP由GGDEF二鸟苷酸环化酶合成并由EAL或HD-GYP磷酸二酯酶水解，每个功能结构域通常附加到不同的感觉模块。HD-GYP结构域蛋白抵抗结构分析，但在这里，我们提出了这个家族的第一个结构代表（1.28 kb），使用不寻常的Bd 1817 HD-GYP蛋白从捕食性细菌噬菌蛭弧菌获得。Bd 1817缺乏大多数HD-GYP家族成员中存在的活性位点酪氨酸，但仍然是其特征的极好模型，与原型RpfG共享48%的序列相似性。蛋白质的结构是高度模块化的，因此提供了一个基础，划定在其他刺激依赖的同系物的域边界。HD-GYP家族中的保守残基围绕双核金属中心聚集，观察到其与磷酸盐分子络合，提供了关于氢氧根离子攻击底物的模式的信息。HD-GYP结构域的折叠和活性位点与EAL蛋白不同，并且对活性位点裂隙的限制性访问指示不同的活性调节模式。包含GYP基序的区域具有新的构象，并且是表面暴露的，并且可用于与结合伴侣（包括GGDEF蛋白）复合。越来越明显的是，许多细菌使用信号分子cyclic-di-GMP来调节各种过程，最值得注意的是，运动性和无柄性之间的转换。重要的是，这种调节对于慢性疾病（粘附，生物膜形成和毒力基因表达）中涉及的几个特征至关重要。通过GGDEF酶合成环状二GMP和通过EAL酶水解的机制已经通过几种晶体结构的分析提出，但是迄今为止没有关于无关的HD-GYP类水解酶的信息。在这里，我们提出了一个不寻常的成员的HD-GYP家庭的捕食性细菌噬菌蛭弧菌的多结构域结构和详细的功能，区分它从更广泛的结构家族的一般HD折叠水解酶。该结构揭示了双核铁中心是如何从几个保守的残基形成的，并为理解HD-GYP家族对c-di-GMP水解的序列要求提供了基础。
Cyclic-di-GMP is a near-ubiquitous bacterial second messenger that is important in localized signal transmission during the control of various processes, including virulence and switching between planktonic and biofilm-based lifestyles. Cyclic-di-GMP is synthesized by GGDEF diguanylate cyclases and hydrolyzed by EAL or HD-GYP phosphodiesterases, with each functional domain often appended to distinct sensory modules. HD-GYP domain proteins have resisted structural analysis, but here we present the first structural representative of this family (1.28 Å), obtained using the unusual Bd1817 HD-GYP protein from the predatory bacterium Bdellovibrio bacteriovorus. Bd1817 lacks the active-site tyrosine present in most HD-GYP family members yet remains an excellent model of their features, sharing 48% sequence similarity with the archetype RpfG. The protein structure is highly modular and thus provides a basis for delineating domain boundaries in other stimulus-dependent homologues. Conserved residues in the HD-GYP family cluster around a binuclear metal center, which is observed complexed to a molecule of phosphate, providing information on the mode of hydroxide ion attack on substrate. The fold and active site of the HD-GYP domain are different from those of EAL proteins, and restricted access to the active-site cleft is indicative of a different mode of activity regulation. The region encompassing the GYP motif has a novel conformation and is surface exposed and available for complexation with binding partners, including GGDEF proteins. It is becoming apparent that many bacteria use the signaling molecule cyclic-di-GMP to regulate a variety of processes, most notably, transitions between motility and sessility. Importantly, this regulation is central to several traits implicated in chronic disease (adhesion, biofilm formation, and virulence gene expression). The mechanisms of cyclic-di-GMP synthesis via GGDEF enzymes and hydrolysis via EAL enzymes have been suggested by the analysis of several crystal structures, but no information has been available to date for the unrelated HD-GYP class of hydrolases. Here we present the multidomain structure of an unusual member of the HD-GYP family from the predatory bacterium Bdellovibrio bacteriovorus and detail the features that distinguish it from the wider structural family of general HD fold hydrolases. The structure reveals how a binuclear iron center is formed from several conserved residues and provides a basis for understanding HD-GYP family sequence requirements for c-di-GMP hydrolysis.