Structure and calcium-binding properties of Frq1, a novel calcium sensor in the yeast Saccharomyces cerevisiae
Structure and calcium-binding properties of Frq1, a novel calcium sensor in the yeast Saccharomyces cerevisiae
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DOI：
10.1021/bi0012890
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发表时间：
2000-10-10
期刊：
影响因子：
2.9
通讯作者：
Thorner, J
中科院分区：
文献类型：
--
作者：
Ames, JB;Hendricks, KB;Thorner, J
FRQ1基因是芽殖酵母生长所必需的，编码190个残基的N-肉豆蔻酰化(MYR)钙结合蛋白。Frq1属于EF-HAND超家族的恢复素/频率蛋白分支，调节酵母磷脂酰肌醇4-激酶亚型。通过核磁共振、荧光和平衡钙结合测量来评估由于N-肉豆蔻酸化和钙结合而引起的Frq1的构象变化。为此，我们在大肠杆菌中表达并纯化了Frq1和MYR-Frq1。在饱和时，Frq1在独立的位置结合了三个钙离子，对应于蛋白质中的第二、第三和第四个EF-Hand基序。第二个点(K-d=10亩M)的亲和力明显弱于第三个和第四个点(K-d=0.4亩M)。MYR Frq1结合Ca~(2+)的K(D)APP为3µM，希尔系数为正(n=1.25)，表明N-肉豆蔻基与Ca~(2+)结合有一定程度的协同作用。Frq1的核磁共振和荧光光谱都显示出很大的依赖于钙的差异，表明钙结合后引起了主要的构象变化。对整个羧基末端结构域(残基K100-I190)进行了几乎完整的序列特异性核磁共振指定。在氨基末端结构域中有20%的残基被指定；未指定的残基显示出非常广泛的核磁共振信号，很可能是由于Frq1二聚作用。钙离子结合的Frq1的核磁共振化学位移和核Overhauser效应(NOE)模式与钙结合的恢复素非常相似，表明Frq1的整体结构与恢复素相似。根据核磁共振数据和与恢复素的同源性，提出了钙离子结合的Frq1的三维结构模型。肉豆蔻酰化对其核磁共振和荧光光谱的影响很小或没有影响，表明肉豆蔻酰基不会显著改变Frq1的结构。相应地，在无钙和结合钙的MYR-Frq1中，肉豆蔻酰基的核磁共振化学位移与游离肉豆蔻酸酯在溶液中的化学位移几乎相同，这表明脂肪酰链是暴露在溶剂中的，而不是隔离在蛋白质的疏水核心中，不同于无钙回收中的肉豆蔻基团。亚细胞分级实验表明，N-肉豆蔻基和钙离子结合均有助于Frq1与膜结合。
The FRQ1 gene is essential for growth of budding yeast and encodes a 190-residue, N-myristoylated (myr) calcium-binding protein. Frq1 belongs to the recoverin/frequenin branch of the EF-hand superfamily and regulates a yeast phosphatidylinositol 4-kinase isoform. Conformational changes in Frq1 due to N-myristoylation and Ca2+ binding were assessed by nuclear magnetic resonance (NMR), fluorescence, and equilibrium Ca2+-binding measurements. For this purpose, Frq1 and myr-Frq1 were expressed in and purified from Escherichia coli. At saturation, Frq1 bound three Ca2+ ions at independent sites, which correspond to the second, third, and fourth EF-hand motifs in the protein. Affinity of the second site (K-d = 10 mu M) was much weaker than that of the third and fourth sites (K-d = 0.4 mu M). Myr Frq1 bound Ca2+ with a K(d)app of 3 mu M and a positive Hill coefficient (n = 1.25), suggesting that the N-myristoyl group confers some degree of cooperativity in Ca2+ binding, as seen previously in recoverin. Both the NMR and fluorescence spectra of Frq1 exhibited very large Ca2+-dependent differences, indicating major conformational changes induced upon Ca2+ binding. Nearly complete sequence-specific NMR assignments were obtained for the entire carboxy-terminal domain (residues K100-I190). Assignments were made for 20% of the residues in the amino-terminal domain; unassigned residues exhibited very broad NMR signals, most likely due to Frq1 dimerization. NMR chemical shifts and nuclear Overhauser effect (NOE) patterns of Ca2+-bound Frq1 were very similar to those of Ca2+-bound recoverin, suggesting that the overall structure of Frq1 resembles that of recoverin. A model of the three-dimensional structure of Ca2+-bound Frq1 is presented based on the NMR data and homology to recoverin. N-myristoylation of Frq1 had little or no effect on its NMR and fluorescence spectra, suggesting that the myristoyl moiety does not significantly alter Frq1 structure. Correspondingly, the NMR chemical shifts for the myristoyl group in both Ca2+-free and Ca2+-bound myr-Frq1 were nearly identical to those of free myristate in solution, indicating that the fatty acyl chain is solvent-exposed and not sequestered within the hydrophobic core of the protein, unlike the myristoyl group in Ca2+-free recoverin. Subcellular fractionation experiments showed that both the N-myristoyl group and Ca2+-binding contribute to the ability of Frq1 to associate with membranes.