Dynamic structure of the calmodulin-binding domain of the plasma membrane Ca-ATPase in native erythrocyte ghost membranes.
Dynamic structure of the calmodulin-binding domain of the plasma membrane Ca-ATPase in native erythrocyte ghost membranes.
复制标题
天然红细胞鬼膜中质膜 Ca-ATP 酶的钙调蛋白结合域的动态结构。
作者：
Y. Yao;J. Gao;T. Squier
我们已经使用频域荧光共振能量转移（FRET）和各向异性测量来鉴定小麦胚芽钙调素（CaM）的结构特性，无论是结合质膜钙- atp酶（pm - ca - atp酶）在天然红细胞鬼膜或肽（C25W），具有相同的序列CaM结合域在pm - ca - atp酶。使用二苯甲酮标记的CaM与使用CaM或pm - ca - atp酶特异性抗体的免疫印迹相结合的交联实验表明，一个CaM分子选择性地结合天然红细胞鬼膜上的pm - ca - atp酶多肽链。在红细胞膜中没有其他高亲和力结合CaM的蛋白质，因此可以测量天然红细胞膜中与pm - ca - atp酶结合的CaM的结构特性。小麦胚芽CaM上位于钙结合环I Cys27处的荧光团芘马来酰亚胺（PMal）和位于钙结合环IV的硝基酪氨酸139之间的FRET测量表明，在CaM与pm - ca - atp酶或C25W结合时，与CaM两个相反的球状结构域相关的平均空间分离和构象异质性几乎相同。结合PM-Ca-ATPase或C25W的pal - cam的溶剂可及性和节段旋转动力学进一步表明，位于Cys27的芘标记周围的局部环境非常相似。然而，CaM与PM-Ca-ATPase结合的整体旋转动力学（phi 2 = 83 +/- 14 ns）比CaM与C25W结合时（phi 2 = 10.3 +/- 0.5 ns）慢得多。这表明CaM与PM-Ca-ATPase的CaM结合域紧密相关，并且所观察到的pyrenylmale亚胺标记CaM的旋转运动是PM-Ca-ATPase上CaM结合域整体运动的特征。CaM与PM-Ca-ATPase或C25W结合的构象异构性和局部环境表明，CaM与Ca-ATPase上类似于C25W的连续氨基酸序列结合，并且与PM-Ca-ATPase内的其他结构元件没有明显的相互作用。与CaM结合pm - ca - atp酶相关的旋转运动速率与流体动力学计算一致，其中位于pm - ca - atp酶羧基端的钙调素结合结构域具有稳定且明确的三级结构，独立于pm - ca - atp酶的其他细胞质结构域。
We have used frequency-domain fluorescence resonance energy transfer (FRET) and anisotropy measurements to identify the structural properties of wheat germ calmodulin (CaM) bound to either the plasma membrane Ca-ATPase (PM-Ca-ATPase) in native erythrocyte ghost membranes or a peptide (C25W) that has an identical sequence to the CaM-binding domain on the PM-Ca-ATPase. Cross-linking experiments using benzophenone labeled CaM in conjunction with immunoblots using antibodies specific for either CaM or the PM-Ca-ATPase indicate that one molecule of CaM selectively binds one PM-Ca-ATPase polypeptide chain in native erythrocyte ghost membranes. There are no other proteins in the erythrocyte membrane that bind CaM with high affinity, permitting the measurement of the structural properties of CaM bound to the PM-Ca-ATPase in native erythrocyte ghost membranes. FRET measurements between the fluorophore pyrene maleimide (PMal) located at Cys27 in calcium binding loop I and nitrotyrosine139 in calcium binding loop IV on wheat germ CaM indicate that the average spatial separation and conformational heterogeneity associated with the two opposing globular domains of CaM are virtually identical upon CaM binding to either the PM-Ca-ATPase or C25W. Measurements of the solvent accessibility and segmental rotational dynamics of PMal-CaM bound to either the PM-Ca-ATPase or C25W further indicate that the local environment around the pyrene label located at Cys27 is very similar. However, the overall rotational dynamics of CaM bound to the PM-Ca-ATPase is much slower (phi 2 = 83 +/- 14 ns) than observed when CaM binds C25W (phi 2 = 10.3 +/- 0.5 ns). This implies that CaM is tightly associated with the CaM-binding domain of the PM-Ca-ATPase and that the observed rotational motion of pyrenylmaleimide labeled CaM is characteristic of the global motion of the CaM-binding domain on the PM-Ca-ATPase. The similar conformational heterogeneity and local environment of CaM bound to either the PM-Ca-ATPase or C25W indicates that CaM binds to a contiguous sequence of amino acids on the Ca-ATPase that are analogous to C25W and that there are no significant interactions with other structural elements within the PM-Ca-ATPase. The rate of rotational motion associated with CaM bound to the PM-Ca-ATPase is consistent with hydrodynamic calculation in which the calmodulin-binding domain located at the carboxyl-terminus of the PM-Ca-ATPase has a stable and defined tertiary structure that is independent of the other cytoplasmic domains of the PM-Ca-ATPase.