Li Jinhui, Xiong Yijia, Bigelow Diana J, Squier Thomas C
Cell Biology Group, Department of Biological Sciences, Fundamental Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
Biochemistry. 2004 Jan 20;43(2):455-63. doi: 10.1021/bi035424v.
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 A and a half-width (HW) of 36 A. 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, where Rav = 14.0 +/- 0.3 A and HW = 3.7 +/- 0.6 A. 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.
诱变和交联测量已经确定了受磷蛋白（PLB）和钙 - ATP酶的胞质序列与跨膜序列之间的特定接触相互作用，并且结合PLB和钙 - ATP酶的高分辨率结构，已被用于构建PLB - ATP酶复合物的模型，这些模型表明PLB在该复合物中采用了更伸展的结构。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共价结合到蛋白脂质体中重构的PLB跨膜和胞质结构域上的发色团之间的平均构象和异质性。在没有钙 - ATP酶的情况下，PLB的胞质结构域相对于跨膜序列呈现出广泛的结构范围，这可以用一个模型来描述，该模型涉及距离的高斯分布，平均距离（Rav）小于21 Å，半高宽（HW）为36 Å。PLB的这种构象异质性与在有机共溶剂中通过核磁共振解析的PLB的C41F突变体的10种结构一致。相比之下，与钙 - ATP酶结合的PLB呈现出独特且高度有序的构象，其中Rav = 14.0±0.3 Å，HW = 3.7±0.6 Å。PLB上结合的发色团之间较小的空间间隔与当前模型中结合的PLB的伸展构象不一致。因此，为了满足PLB和钙 - ATP酶已知的相互作用位点，这些发现表明钙 - ATP酶的核苷酸结合结构域重新定向至双层表面，以使已知的PLB结合位点与PLB氨基末端附近的残基紧密并列。PLB结合诱导钙 - ATP酶核苷酸结合结构域构象改变被认为是与PLB抑制泵相关的钙敏感性降低的基础。
