Application of Synthetic Peptide Arrays To Uncover Cyclic Di-GMP Binding Motifs
Application of Synthetic Peptide Arrays To Uncover Cyclic Di-GMP Binding Motifs
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DOI：
10.1128/jb.00377-15
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发表时间：
2016-01-01
影响因子：
3.2
通讯作者：
Haeussler, Susanne
中科院分区：
文献类型：
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作者：
Duevel, Juliane;Bense, Sarina;Haeussler, Susanne
高水平的通用细菌第二信使环二GMP（c-di-GMP）促进许多细菌表面附着生长的建立。c-di-GMP不仅可以与核酸结合并直接控制基因表达，而且还可以与各种具有特殊功能的蛋白质结合并协调其活性。自20世纪90年代初发展以来，合成肽阵列技术已成为高通量方法的有力工具，并成功应用于研究蛋白质-配体相互作用的结合特异性。在这项研究中，我们使用肽阵列来揭示铜绿假单胞菌蛋白（PA 3740）的c-di-GMP结合位点，该蛋白是在化学蛋白质组学方法中分离的。PA 3740显示出以高亲和力结合c-di-GMP，并且肽阵列发现LKKALKKQTNLR是推定的c-diGMP结合基序。最有趣的是，与先前鉴定的PilZ结构域的c-di-GMP结合基序（RXXXR）或二鸟苷酸环化酶的I位点（RXXD）不同，两个亮氨酸残基和一个谷氨酰胺残基而不是带电荷的氨基酸提供了结合序列的关键残基。这三个氨基酸是高度保守的整个PA 3740同系物，和他们的单一交换丙氨酸减少c-di-GMP结合内的全长protein.IMPORTANCEIn许多细菌病原体的通用细菌第二信使c-di-GMP管理开关从无菌的，能动的生长模式的固着，生物膜生长模式。细菌使其细胞内c-di-GMP水平适应各种环境挑战。几类c-di-GMP结合蛋白的结构特征已被确定，并已鉴定出不同的c-di-GMP结合结构域。然而，对于几种c-di-GMP受体，结合基序仍有待确定。在这里，我们表明，使用的合成肽阵列允许的c-di-GMP结合基序的一个假定的c-di-GMP受体蛋白的机会致病菌铜绿假单胞菌的鉴定。合成肽阵列的应用将有助于寻找额外的c-di-GMP受体蛋白，并有助于c-di-GMP结合基序的表征。
High levels of the universal bacterial second messenger cyclic di-GMP (c-di-GMP) promote the establishment of surface-attached growth in many bacteria. Not only can c-di-GMP bind to nucleic acids and directly control gene expression, but it also binds to a diverse array of proteins of specialized functions and orchestrates their activity. Since its development in the early 1990s, the synthetic peptide array technique has become a powerful tool for high-throughput approaches and was successfully applied to investigate the binding specificity of protein-ligand interactions. In this study, we used peptide arrays to uncover the c-di-GMP binding site of a Pseudomonas aeruginosa protein (PA3740) that was isolated in a chemical proteomics approach. PA3740 was shown to bind c-di-GMP with a high affinity, and peptide arrays uncovered LKKALKKQTNLR to be a putative c-diGMP binding motif. Most interestingly, different from the previously identified c-di-GMP binding motif of the PilZ domain (RXXXR) or the I site of diguanylate cyclases (RXXD), two leucine residues and a glutamine residue and not the charged amino acids provided the key residues of the binding sequence. Those three amino acids are highly conserved across PA3740 homologs, and their singular exchange to alanine reduced c-di-GMP binding within the full-length protein.IMPORTANCEIn many bacterial pathogens the universal bacterial second messenger c-di-GMP governs the switch from the planktonic, motile mode of growth to the sessile, biofilm mode of growth. Bacteria adapt their intracellular c-di-GMP levels to a variety of environmental challenges. Several classes of c-di-GMP binding proteins have been structurally characterized, and diverse c-di-GMP binding domains have been identified. Nevertheless, for several c-di-GMP receptors, the binding motif remains to be determined. Here we show that the use of a synthetic peptide array allowed the identification of a c-di-GMP binding motif of a putative c-di-GMP receptor protein in the opportunistic pathogen P. aeruginosa. The application of synthetic peptide arrays will facilitate the search for additional c-di-GMP receptor proteins and aid in the characterization of c-di-GMP binding motifs.