Essential role for Pro21 in phospholamban for optimal inhibition of the Ca-ATPase.
Essential role for Pro21 in phospholamban for optimal inhibition of the Ca-ATPase.
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Pro21 在受磷蛋白中发挥着重要作用，可实现 Ca-ATP 酶的最佳抑制。
DOI：
10.1021/bi051075o
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Bigelow,DianaJ
中科院分区：
文献类型：
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作者：
Li,Jinhui;Boschek,CurtB;Xiong,Yijia;Sacksteder,ColetteA;Squier,ThomasC;Bigelow,DianaJ
我们已经研究了柔性铰链区的功能作用，该柔性铰链区位于序列TIEMP 21附近，该序列连接受磷蛋白（PLB）的N-末端胞质和C-末端跨膜螺旋结构域。具体来说，我们问，如果这个区域的构象是重要的，以达到最佳的抑制作用与钙-ATP酶。构建基因工程化的PLB突变体，其中Pro21突变为丙氨酸（P21 A-PLBC）;在该构建体中，所有三个跨膜半胱氨酸被丙氨酸取代以稳定PLB的单体形式，并且在铰链元件（A24 C）附近的位置24处引入独特的半胱氨酸，允许荧光素-5-马来酰亚胺（FMal）的位点特异性附着以监测结构变化。与先前在心脏SR微粒体中的测量结果一致，当与PLBC（Pro 21）共重构时，由于与钙依赖性结构转变相关的协同性降低，与Ca-ATP酶的半数最大激活（Ca 1/2）相关的钙浓度（290 ± 10 nM）变为580 ± 20 nM。动力学模拟表明，PLBC与Ca-ATP酶的结果在一个75%的减少与第二个高亲和力的钙结合位点的形成相关的平衡常数。相比之下，在用P21 A-PLBC重建Ca-ATP酶时，KCa 1/2降低43%，这可以通过将与钙依赖性结构活化相关的平衡常数降低50%来模拟。P21 A-PLBC的抑制作用减弱与铰链元件结构的改变有关，如FMal相对于天然结构的溶剂可及性减弱所证明的。同样，使用圆二色谱和荧光光谱观察到P21 A-PLBCare的α-螺旋含量增加和羧基末端结构域的迁移率降低。总的来说，这些结果表明，通过P21 A-PLBC突变后二级结构元件的稳定化，PLB的羧基末端结构域的总体尺寸增加，这导致PLB的氨基末端胞质部分有效抑制Ca-ATP酶的能力降低。此外，这些结果表明，PLB中的柔性铰链区的非结构化特征是至关重要的最佳抑制与Ca-ATP酶的相互作用，并建议其作为构象开关的作用。
We have investigated the functional role of the flexible hinge region centered near the sequence TIEMP21, which connects the N-terminal cytosolic and C-terminal membrane-spanning helical domains of phospholamban (PLB). Specifically, we ask if the conformation of this region is important to attain optimal inhibitory interactions with the Ca-ATPase. A genetically engineered PLB mutant was constructed in which Pro21was mutated to an alanine (P21A-PLBC); in this construct, all three transmembrane cysteines were substituted with alanines to stabilize the monomeric form of PLB, and a unique cysteine was introduced at position 24 near the hinge element (A24C), permitting the site-specific attachment of fluorescein-5-maleimide (FMal) to monitor structure changes. In agreement with prior measurements in cardiac SR microsomes, the calcium concentration associated with half-maximal activation (Ca1/2) of the Ca-ATPase, 290 ± 10 nM, is shifted to 580 ± 20 nM when co-reconstituted with PLBC(Pro21) as a result of a reduction in the cooperativity associated with the calcium-dependent structural transition. Kinetic simulations indicate that PLBCassociation with the Ca-ATPase results in a 75% reduction in the equilibrium constant associated with the formation of the second high-affinity calcium binding site. In comparison, there is a 43% reduction in KCa1/2upon reconstitution of the Ca-ATPase with P21A-PLBC, which can be simulated by decreasing the equilibrium constant associated with the calcium-dependent structural activation by 50%. The diminished inhibitory action of P21A-PLBCis associated with alterations in the structure of the hinge element, as evidenced by the diminished solvent accessibility of FMal relative to the native structure. Likewise, increases in the α-helical content and decreases in the mobility of the carboxyl-terminal domain of P21A-PLBCare observed using circular dichroism and fluorescence spectroscopy. Collectively, these results indicate that the overall dimensions of the carboxyl-terminal domain of PLB are increased through a stabilization of secondary structural elements upon mutation in P21A-PLBCthat result in a reduction in the ability of the amino-terminal cytosolic portion of PLB to productively inhibit the Ca-ATPase. Further, these results suggest that the unstructured characteristics of the flexible hinge region in PLB are critical for optimal inhibitory interactions with the Ca-ATPase and suggest its role as a conformational switch.