Phosphorylation by cAMP-dependent protein kinase modulates the structural coupling between the transmembrane and cytosolic domains of phospholamban.
Phosphorylation by cAMP-dependent protein kinase modulates the structural coupling between the transmembrane and cytosolic domains of phospholamban.
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cAMP 依赖性蛋白激酶的磷酸化可调节受磷蛋白的跨膜结构域和胞质结构域之间的结构耦合。
DOI：
10.1021/bi034708c
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Squier,ThomasC
中科院分区：
文献类型：
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作者：
Li,Jinhui;Bigelow,DianaJ;Squier,ThomasC
我们已经使用频域荧光光谱法来研究调节蛋白受磷蛋白（PLB）的跨膜和胞质结构域之间的结构联系。使用具有用荧光团N-（1-芘基）马来酰亚胺（PMal）衍生的单个半胱氨酸（Cys 24）的工程化PLB，我们已经使用荧光共振能量转移（FRET）来测量在PLB的氨基末端附近的跨膜结构域中结合到Cys 24的PMal和胞质结构域中的Tyr 6之间的平均空间分离和构象异质性。在这些测量中，PMal用作FRET供体，Tyr 6在其被四硝基甲烷硝化后用作FRET受体。PLB的天然结构在定点诱变和化学修饰后得以保留，如衍生化的PLB在共重构后完全调节Ca-ATP酶的能力所示。为了评估磷酸化如何调节PLB本身的结构，在由提取的肌浆网膜脂质制成的膜囊泡中重构PLB后进行FRET测量。我们发现，胞质结构域的PLB假设一个广泛的构象相对于跨膜序列，与其他结构数据一致，表明存在一个灵活的铰链区之间的跨膜和胞质结构域的PLB。PKA对Ser 16的磷酸化使PMal的Cys 24和nitroTyr 6之间的空间间隔减小了3倍，构象异质性降低了近2倍，这表明PLB铰链区的稳定性可能是通过磷酸化Ser 16和Arg 13之间的静电连接促进了卷曲到螺旋的转变。这种结构转变有可能充当构象开关，因为Ca-ATP酶的抑制需要破坏铰链元件附近PLB的二级结构，以允许与位于膜表面上方约50 Å的位点处的核苷酸结合结构域结合。磷酸化后，铰链结构域中螺旋内容物的稳定化将通过降低PLB胞质结构域的最大尺寸来破坏这种抑制性相互作用。因此，Ser 16磷酸化后PLB结构的稳定是开关机制的一部分，其功能是改变PLB和调节酶抑制的Ca-ATP酶的核苷酸结合结构域之间的结合相互作用。
We have used frequency-domain fluorescence spectroscopy to investigate the structural linkage between the transmembrane and cytosolic domains of the regulatory protein phospholamban (PLB). Using an engineered PLB having a single cysteine (Cys24) derivatized with the fluorophoreN-(1-pyrenyl)maleimide (PMal), we have used fluorescence resonance energy transfer (FRET) to measure the average spatial separation and conformational heterogeneity between PMal bound to Cys24in the transmembrane domain and Tyr6in the cytosolic domain near the amino terminus of PLB. In these measurements, PMal serves as a FRET donor, and Tyr6serves as a FRET acceptor following its nitration by tetranitromethane. The native structure of PLB is retained following site-directed mutagenesis and chemical modification, as indicated by the ability of the derivatized PLB to fully regulate the Ca-ATPase following their co-reconstitution. To assess how phosphorylation modulates the structure of PLB itself, FRET measurements were made following reconstitution of PLB in membrane vesicles made from extracted sarcoplasmic reticulum membrane lipids. We find that the cytosolic domain of PLB assumes a wide range of conformations relative to the transmembrane sequence, consistent with other structural data indicating the presence of a flexible hinge region between the transmembrane and cytosolic domains of PLB. Phosphorylation of Ser16by PKA results in a 3 Å decrease in the spatial separation between PMal at Cys24and nitroTyr6and an almost 2-fold decrease in conformational heterogeneity, suggesting a stabilization of the hinge region of PLB possibly through an electrostatic linkage between phosphoSer16and Arg13that promotes a coil-to-helix transition. This structural transition has the potential to function as a conformational switch, since inhibition of the Ca-ATPase requires disruption of the secondary structure of PLB in the vicinity of the hinge element to permit association with the nucleotide binding domain at a site located approximately 50 Å above the membrane surface. Following phosphorylation, the stabilization of the helical content in the hinge domain will disrupt this inhibitory interaction by reducing the maximal dimension of the cytosolic domain of PLB. Thus, stabilization of the structure of PLB following phosphorylation of Ser16is part of a switching mechanism, which functions to alter binding interactions between PLB and the nucleotide binding domain of the Ca-ATPase that modulates enzyme inhibition.