首页> 外文期刊>Protein engineering design & selection: PEDS >Design and characterization of a soluble fragment of the extracellular ligand-binding domain of the peptide hormone receptor guanylyl cyclase-C.
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Design and characterization of a soluble fragment of the extracellular ligand-binding domain of the peptide hormone receptor guanylyl cyclase-C.

机译:肽激素受体鸟苷酸环化酶-C的细胞外配体结合域的可溶性片段的设计和表征。

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摘要

The intestinal guanylyl cyclase-C (GC-C) was originally identified as an Escherichia coli heat-stable enterotoxin (STa) receptor. STa stimulates GC-C to much higher activity than the endogenous ligands guanylin and uroguanylin, causing severe diarrhea. To investigate the interactions of the endogenous and bacterial ligands with GC-C, we designed and characterized a soluble and properly folded fragment of the extracellular ligand-binding domain of GC-C. The membrane-bound guanylyl cyclases exhibit a single transmembrane spanning helix and a globularly folded extracellular ligand-binding domain that comprises about 410 of 1050 residues. Based on the crystal structure of the dimerized-binding domain of the guanylyl cyclase-coupled atrial natriuretic peptide receptor and a secondary structure-guided sequence alignment, we generated a model of the extracellular domain of GC-C comprised of two subdomains. Mapping of mutational and cross-link data onto this structural model restricts the ligand-binding regionto the membrane proximal subdomain. We thus designed miniGC-C, a 197 amino acid fragment that mimics the ligand-binding membrane proximal subdomain. Cloning, expression and spectroscopic studies reveal miniGC-C to be a soluble and properly folded protein with a distinct secondary and tertiary structure. MiniGC-C binds STa with nanomolar affinity.
机译:肠道鸟苷基环化酶-C(GC-C)最初被鉴定为大肠杆菌的热稳定肠毒素(STa)受体。 STa刺激GC-C的活性比内源性鸟苷和尿鸟苷高得多,从而引起严重的腹泻。为了研究内源和细菌配体与GC-C的相互作用,我们设计并表征了GC-C胞外配体结合域的可溶性和正确折叠的片段。膜结合的鸟苷酰环化酶表现出单个跨膜的螺旋结构和球状折叠的细胞外配体结合结构域,其包含1050个残基中的约410个。基于鸟苷酸环化酶偶联的心钠素受体的二聚化结合结构域的晶体结构和二级结构指导的序列比对,我们生成了由两个亚结构域组成的GC-C细胞外结构域模型。突变和交联数据到该结构模型的映射将配体结合区域限制在膜近端亚结构域。因此,我们设计了miniGC-C,它是一个197个氨基酸的片段,可模拟配体结合膜近端亚结构域。克隆,表达和光谱学研究表明miniGC-C是一种可溶性且正确折叠的蛋白质,具有独特的二级和三级结构。 MiniGC-C以纳摩尔亲和力结合STa。

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