A flagellum-specific calcium sensor
A flagellum-specific calcium sensor
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DOI：
10.1074/jbc.m505777200
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发表时间：
2005-12-02
影响因子：
4.8
通讯作者：
Engman, DM
中科院分区：
文献类型：
--
作者：
Buchanan, KT;Ames, JB;Engman, DM
鞭毛钙结合蛋白（FCaBP）的鞭毛原生动物克氏锥虫协会通过其N-末端肉豆蔻酸和棕榈酸部分的鞭毛膜钙调节，构象依赖的方式。这种定位机制类似于神经元钙传感器所描述的机制，神经元钙传感器的构象发生钙依赖性变化，从而调节膜相互作用和伴侣结合的酰基的可用性。为了测试FCaBP是否经历钙依赖性构象变化，并探索这种变化在鞭毛靶向中的作用，我们首先将点突变引入到FCaBP的两个EF-手钙结合位点中的每一个中以定义它们的亲和力。对重组EF-3突变体（E151 Q）、EF-4突变体（E188 Q）和双突变体蛋白的分析表明，EF-3是高亲和力位点（Kd类似于9 μ M），EF-4是低亲和力位点（Kd类似于120 μ M）。这些分配也与部分（E188 Q），几乎完全（E151 Q），和完全（E151 Q，E188 Q）破坏钙诱导的构象变化，通过NMR光谱测定。我们接下来在T.克氏上鞭毛体。这些转蛋白定位于鞭毛，这表明存在的钙依赖性相互作用的FCaBP是独立的其内在的钙结合能力。通过FCaBP亲和层析鉴定了几种蛋白质，其以钙依赖性方式与FCaBP相互作用，但对FCaBP的钙结合具有差异依赖性。这些发现可能对蛋白质调节的钙酰基转换机制具有更广泛的意义。
The flagellar calcium-binding protein ( FCaBP) of the flagellated protozoan Trypanosoma cruzi associates with the flagellar membrane via its N-terminal myristate and palmitate moieties in a calcium-modulated, conformation-dependent manner. This mechanism of localization is similar to that described for neuronal calcium sensors, which undergo calcium-dependent changes in conformation, which modulate the availability of the acyl groups for membrane interaction and partner association. To test whether FCaBP undergoes a calcium-dependent conformational change and to explore the role of such a change in flagellar targeting, we first introduced point mutations into each of the two EF-hand calcium-binding sites of FCaBP to define their affinities. Analysis of recombinant EF-3 mutant ( E151Q), EF-4 mutant ( E188Q), and double mutant proteins showed EF-3 to be the high affinity site ( K-d similar to 9 mu M) and EF-4 the low affinity site ( K-d similar to 120 mu M). These assignments also correlated with partial ( E188Q), nearly complete ( E151Q), and complete ( E151Q, E188Q) disruption of calcium-induced conformational changes determined by NMR spectrometry. We next expressed the FCaBP E151Q mutant and the double mutant in T. cruzi epimastigotes. These transproteins localized to the flagellum, suggesting the existence of a calcium-dependent interaction of FCaBP that is independent of its intrinsic calcium binding capacity. Several proteins were identified by FCaBP affinity chromatography that interact with FCaBP in a calcium-dependent manner, but with differential dependence on calcium-binding by FCaBP. These findings may have broader implications for the calcium acyl switch mechanism of protein regulation.