Changes in biological activity and folding of guanylate cyclase-activating protein 1 as a function of calcium
Changes in biological activity and folding of guanylate cyclase-activating protein 1 as a function of calcium
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DOI：
10.1021/bi972306x
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发表时间：
1998-01-06
期刊：
影响因子：
2.9
通讯作者：
Palczewski, K
中科院分区：
文献类型：
--
作者：
Rudnicka-Nawrot, M;Surgucheva, I;Palczewski, K
鸟苷酸环化酶激活蛋白 1 (GCAP1) 是一种光感受器特异性 Ca2+ 结合蛋白：在光转导的恢复阶段激活视网膜鸟苷酸环化酶 1 (GC1)。与钙调蛋白超家族的其他 Ca2+ 结合蛋白相比，GCAP1 的 Ca2+ 游离形式刺激效应酶。在本研究中，我们通过有限的蛋白水解和诱变分析了 GCAP1 结构中 Ca2+ 依赖性变化，以了解 GC1 活性的 Ca2+ 敏感调节机制。GCAP1 从 Ca2+ 结合形式到 Ca2+ 游离形式的变化增加了 Ca2+ 游离形式的敏感性GCAP1被胰蛋白酶水解，测序数据显示，在Ca2+结合形式中，只有GCAP1的N端（肉豆蔻酰化Gly(2)-Lys(9)）和C端（171-205片段）被胰蛋白酶去除，而在Ca2+游离形式中，GCAP1很容易降解成小片段。通过定点诱变连续灭活每个功能性 EF 环表明，只有 EF3 和 EF4 有助于 Ca2+ 依赖性 GCAP1 灭活。 GCAP1((ED)-D-75,(ED)-D-111,(ED)-D-155)突变体不结合Ca2+并以[Ca2+]独立的方式刺激GC1。 GCAP1 和 GCAP2，但不是 S-100 beta，GC1 的高 [Ca2+]（游离）激活剂，与高 [Ca2+]（游离）的三重突变体竞争，以相似的 IC50 抑制 GC1。这些竞争结果与 GC1 和 GCAP 之间的相似亲和力一致。我们的数据表明，GCAP1 在 Ca2+ 结合过程中经历了主要的构象变化，并且 EF3 和 EF4 基序负责 GCAP1 结构的变化，从而将该蛋白从 GC1 的激活剂转化为抑制剂。
Guanylate cyclase-activating protein 1 (GCAP1), a photoreceptor-specific Ca2+-binding protein: activates retinal guanylate cyclase 1 (GC1) during the recovery phase of phototransduction. In contrast to other Ca2+-binding proteins from the calmodulin superfamily, the Ca2+-free form of GCAP1 stimulates the effector enzyme, Ln this study, we analyzed the Ca2+-dependent changes in GCAP1 structure by limited proteolysis and mutagenesis in order to understand the mechanism of Ca2+-sensitive modulation of GC1 activity, The change from a Ca2+-bound to a Ca2+-free form of GCAP1 increased susceptibility of Ca2+-free GCAP1 to proteolysis by trypsin, Sequencing data revealed that in the Ca2+-bound form, only the N-terminus (myristoylated Gly(2)-Lys(9)) and C-terminus (171-205 fragment) of GCAP1 are removed by trypsin, while in the Ca2+-free form, GCAP1 is readily degraded to small fragments. Successive inactivation of each of the functional EF loops by site-directed mutagenesis showed that only EF3 and EF4 contribute to a Ca2+-dependent inactivation of GCAP1. GCAP1((ED)-D-75,(ED)-D-111,(ED)-D-155) mutant did not bind Ca2+ and stimulated GC1 in a [Ca2+]-independent manner. GCAP1 and GCAP2, but not S-100 beta, a high [Ca2+](free) activator of GC1, competed with the triple mutant at high [Ca2+](free), inhibiting GC1 with similar IC50's. These competition results are consistent with comparable affinities between GC1 and GCAPs, Our data suggest that GCAP1 undergoes major conformational changes during Ca2+ binding and that EF3 and EF4 motifs are responsible for changes in the GCAP1 structure that converts this protein from the activator to the inhibitor of GC1.