Phospholamban binds in a compact and ordered conformation to the Ca-ATPase.
Phospholamban binds in a compact and ordered conformation to the Ca-ATPase.
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Phospholamban 以紧凑且有序的构象与 Ca-ATP 酶结合。
DOI：
10.1021/bi035424v
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发表时间：
2004
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;Xiong,Yijia;Bigelow,DianaJ;Squier,ThomasC
诱变和交联测量已经确定了磷蛋白（PLB）和ca - atp酶的细胞质和跨膜序列之间的特定接触相互作用，并结合PLB和ca - atp酶的高分辨率结构，已用于构建PLB- atp酶复合物的模型，这表明PLB在该复合物中采用了更扩展的结构。为了直接验证这些预测，我们使用荧光共振能量转移来测量在蛋白脂质体中重组的PLB的跨膜和细胞质结构域共价结合的发色团之间的平均构象和异质性。在缺乏ca - atp酶的情况下，相对于跨膜序列，PLB的细胞质结构域具有广泛的结构，可以使用涉及距离高斯分布的模型来描述，平均距离（Rav）小于21 Å，半宽度（HW）为36 Å。PLB的这种构象非均质性与有机共溶剂中PLB的C41F突变体的核磁共振解析的10种结构一致。相比之下，与ca - atp酶结合的PLB具有独特且高度有序的构象，其中erav = 14.0±0.3 Å， HW = 3.7±0.6 Å。PLB上结合发色团之间的小空间间隔与当前模型中结合PLB的扩展构象不一致。因此，为了满足已知的PLB和ca - atp酶的相互作用位点，这些研究结果表明，ca - atp酶的核苷酸结合域向双层表面重新定向，使已知的PLB结合位点与PLB的氨基末端附近的残基紧密并置。通过PLB结合诱导ca - atp酶核苷酸结合域构象的改变被认为是与PLB抑制泵相关的钙敏感性降低的基础。
Mutagenesis and cross-linking measurements have identified specific contact interactions between the cytosolic and the transmembrane sequences of phospholamban (PLB) and the Ca-ATPase, and in conjunction with the high-resolution structures of PLB and the Ca-ATPase, have been used to construct models of the PLB-ATPase complex, which suggest that PLB adopts a more extended structure within this complex. To directly test these predictions, we have used fluorescence resonance energy transfer to measure the average conformation and heterogeneity between chromophores covalently bound to the transmembrane and cytosolic domains of PLB reconstituted in proteoliposomes. In the absence of the Ca-ATPase, the cytosolic domain of PLB assumes a wide range of structures relative to the transmembrane sequence, which can be described using a model involving a Gaussian distribution of distances with an average distance (Rav) of less than 21 Å and a half-width (HW) of 36 Å. This conformational heterogeneity of PLB is consistent with the 10 structures resolved by NMR for the C41F mutant of PLB in organic cosolvents. In contrast, PLB bound to the Ca-ATPase assumes a unique and highly ordered conformation, whereRav= 14.0 ± 0.3 Å and HW = 3.7 ± 0.6 Å. The small spatial separation between the bound chromophores on PLB is inconsistent with an extended conformation of bound PLB in current models. Thus, to satisfy known interaction sites of PLB and the Ca-ATPase, these findings suggest a reorientation of the nucleotide binding domain of the Ca-ATPase toward the bilayer surface to bring known PLB binding sites into close juxtaposition with residues near the amino-terminus of PLB. Induction of an altered conformation of the nucleotide binding domain of the Ca-ATPase by PLB binding is suggested to underlie the reduced calcium sensitivity associated with PLB inhibition of the pump.