Differential ligand-selective control of opposing enzymatic activities within a bifunctional c-di-GMP enzyme
Differential ligand-selective control of opposing enzymatic activities within a bifunctional c-di-GMP enzyme
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DOI：
10.1073/pnas.2100657118
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发表时间：
2021-09
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影响因子：
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通讯作者：
Dayna C. Patterson;Myrrh Perez Ruiz;H. Yoon;Johnnie A. Walker;J. Armache;N. Yennawar;Emily E. Weinert
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作者：
Dayna C. Patterson;Myrrh Perez Ruiz;H. Yoon;Johnnie A. Walker;J. Armache;N. Yennawar;Emily E. Weinert
双功能酶广泛分布于细菌中，参与调节细菌表型;然而，控制相反输出结构域活性的调节机制仍然难以捉摸。DcpG的研究表明，传感器珠蛋白结构域的配体的结合差异影响GGDEF和EAL域的活动，并强调蛋白质构象变化在调节酶活性的作用。不寻常的传感器珠蛋白域的特点，包括血红素中点电位，可能是重要的独特的监管性质的DcpG。由于树状类芽孢杆菌通过调节生物膜形成来响应气体环境的变化，DcpG可能在调节对O2和NO水平变化的生理响应中很重要，从而确定细菌中血红素传感器信号传导的作用。环二聚鸟苷一磷酸（c-di-GMP）作为第二信使调节细菌细胞过程，包括生物膜的形成。虽然在细菌基因组中广泛预测含有c-di-GMP合成（GGDEF）和c-di-GMP水解（EAL）结构域的蛋白质，但对具有相反酶活性的结构域如何在单个多肽内调节知之甚少。在此，我们报告了一个球蛋白偶联传感器蛋白（GCS）从类芽孢杆菌dendritiformis（DcpG）的双功能c-di-GMP酶活性的表征。DcpG含有一个调节传感器球蛋白结构域连接到二鸟苷酸环化酶（GGDEF）和磷酸二酯酶（EAL）结构域，其通过与血红素结合的气体差异调节; GGDEF结构域活性由球蛋白结构域的Fe（II）-NO状态激活，而EAL结构域活性由Fe（II）-O2状态激活。DcpG的体外活性在体内通过响应于气体环境的树状假单胞菌的生物膜形成来模拟，其中一氧化氮条件导致最大量的生物膜形成。DcpG响应于配体结合而差异控制GGDEF和EAL结构域活性的能力可能是由于珠蛋白结构域的不寻常性质，包括快速配体解离速率和高中点电位。利用小角X射线散射和负染电子显微镜研究的结构信息，我们开发了一个结构模型的DcpG，提供有关的监管机制的信息。这些研究提供了有关全长GCS蛋白结构的信息，并深入了解了单个调控结构域可以选择性地控制具有相反酶活性的输出结构域的机制。
Significance Bifunctional enzymes are widely distributed throughout bacteria and are involved in modulating bacterial phenotypes; however, regulatory mechanisms that control the activities of the opposing output domains have remained elusive. Studies on DcpG demonstrate that binding of ligands to the sensor globin domain differentially affect GGDEF and EAL domain activities and highlight a role for protein conformational changes in modulating enzymatic activity. Unusual sensor globin domain characteristics, including heme midpoint potentials, are likely important for the unique regulatory properties of DcpG. As Paenibacillus dendritiformis responds to changes in the gaseous environment by modulating biofilm formation, DcpG is likely important in modulating physiological responses to changes in O2 and NO levels, identifying a role for heme sensor signaling in the bacterium. Cyclic dimeric guanosine monophosphate (c-di-GMP) serves as a second messenger that modulates bacterial cellular processes, including biofilm formation. While proteins containing both c-di-GMP synthesizing (GGDEF) and c-di-GMP hydrolyzing (EAL) domains are widely predicted in bacterial genomes, it is poorly understood how domains with opposing enzymatic activity are regulated within a single polypeptide. Herein, we report the characterization of a globin-coupled sensor protein (GCS) from Paenibacillus dendritiformis (DcpG) with bifunctional c-di-GMP enzymatic activity. DcpG contains a regulatory sensor globin domain linked to diguanylate cyclase (GGDEF) and phosphodiesterase (EAL) domains that are differentially regulated by gas binding to the heme; GGDEF domain activity is activated by the Fe(II)-NO state of the globin domain, while EAL domain activity is activated by the Fe(II)-O2 state. The in vitro activity of DcpG is mimicked in vivo by the biofilm formation of P. dendritiformis in response to gaseous environment, with nitric oxide conditions leading to the greatest amount of biofilm formation. The ability of DcpG to differentially control GGDEF and EAL domain activity in response to ligand binding is likely due to the unusual properties of the globin domain, including rapid ligand dissociation rates and high midpoint potentials. Using structural information from small-angle X-ray scattering and negative stain electron microscopy studies, we developed a structural model of DcpG, providing information about the regulatory mechanism. These studies provide information about full-length GCS protein architecture and insight into the mechanism by which a single regulatory domain can selectively control output domains with opposing enzymatic activities.