Structure and monomer/dimer equilibrium for the guanylyl cyclase domain of the optogenetics protein RhoGC
Structure and monomer/dimer equilibrium for the guanylyl cyclase domain of the optogenetics protein RhoGC
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DOI：
10.1074/jbc.m117.812685
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发表时间：
2017-12-29
影响因子：
4.8
通讯作者：
Oprian, Daniel D.
中科院分区：
文献类型：
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作者：
Kumar, Ramasamy P.;Morehouse, Benjamin R.;Oprian, Daniel D.
RhoGC是一种融合蛋白，来自水生真菌芽枝菌（Blastocladiella emersonii），它结合了1型视紫红质结构域和一个冠酰环化酶结构域。作为操纵环核苷酸信号通路的光遗传学工具，它已经引起了人们的兴奋。为了研究环化酶活性的调控，我们从大肠杆菌中分离出含（GCwCC(Rho)）和不含（GC(Rho)）的环化酶结构域。这两种结构都具有组成活性，但通过尺寸排除色谱和分析性超离心确定为单体，而其他III类核苷酸环化酶是功能性二聚体。我们还观察到GC（Rho）晶体在不对称单元中只有一个单体。当晶体在不可环化的底物类似物2,3-二脱氧鸟苷-5-三磷酸、MnCl2和酒石酸盐的存在下生长时形成二聚体，但它们的四级结构不符合III类酶的典型配对。此外，该结构含有由活性位点Cys残基形成的二硫键。我们认为二硫化物不太可能在细胞内还原条件下形成，这就提出了这种不寻常的二聚体可能在全长RhoGC的调控中具有生物学相关作用的可能性。虽然我们没有用直接的方法观察，但根据活性对总酶浓度的依赖，我们确定了一个功能二聚体为活性状态。GC（Rho）单体的低亲和力在这类酶中是不寻常的，这表明二聚体的形成可能有助于全长蛋白的光激活。
RhoGC is a fusion protein from the aquatic fungus Blastocladiella emersonii, combining a type I rhodopsin domain with a guanylyl cyclase domain. It has generated excitement as an optogenetics tool for the manipulation of cyclic nucleotide signaling pathways. To investigate the regulation of the cyclase activity, we isolated the guanylyl cyclase domain from Escherichia coli with (GCwCC(Rho)) and without (GC(Rho)) the coiled-coil linker. Both constructs were constitutively active but were monomeric as determined by size-exclusion chromatography and analytical ultracentrifugation, whereas other class III nucleotidyl cyclases are functional dimers. We also observed that crystals of GC(Rho) have only a monomer in an asymmetric unit. Dimers formed when crystals were grown in the presence of the non-cyclizable substrate analog 2,3-dideoxyguanosine-5-triphosphate, MnCl2, and tartrate, but their quaternary structure did not conform to the canonical pairing expected for class III enzymes. Moreover, the structure contained a disulfide bond formed with an active-site Cys residue required for activity. We consider it unlikely that the disulfide would form under intracellular reducing conditions, raising the possibility that this unusual dimer might have a biologically relevant role in the regulation of full-length RhoGC. Although we did not observe it with direct methods, a functional dimer was identified as the active state by following the dependence of activity on total enzyme concentration. The low affinity observed for GC(Rho) monomers is unusual for this enzyme class and suggests that dimer formation may contribute to light activation of the full-length protein.