STRUCTURAL MODEL OF THE PHOSPHOLAMBAN ION-CHANNEL COMPLEX IN PHOSPHOLIPID-MEMBRANES
STRUCTURAL MODEL OF THE PHOSPHOLAMBAN ION-CHANNEL COMPLEX IN PHOSPHOLIPID-MEMBRANES
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DOI：
10.1006/jmbi.1995.0263
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发表时间：
1995-05-12
影响因子：
5.6
通讯作者：
SMITH, SO
中科院分区：
文献类型：
--
作者：
ARKIN, IT;ROTHMAN, M;SMITH, SO
Phospholamban 是一种 52 个氨基酸残基的膜蛋白，参与心肌细胞肌浆网膜钙水平的调节。该蛋白的 N 端 30 个氨基酸残基大部分是亲水性的，并且包括两个位点，其磷酸化被认为可以解离受磷蛋白和 Ca2+ ATP 酶之间的抑制复合物。 C 端 22 个氨基酸残基大部分是疏水性的，将蛋白质锚定在膜上，并负责 Ca2+ 选择性离子电导。跨膜结构域之间的特异性相互作用稳定了五聚体蛋白质复合物。我们获得了全长蛋白质的圆二色性 (CD)、透射傅里叶变换红外 (FTIR) 和衰减全反射傅里叶变换红外 (ATR-FTIR) 光谱，并将这些结果与包含跨膜结构域的 28 个残基肽的结果进行了比较。通过 CD 和 FTIR 检测，重组到磷脂膜中的两种蛋白质大部分都是 α 螺旋。偏振 ATR-FTIR 测量表明，胞质螺旋和跨膜螺旋均垂直于膜平面，相对于膜法线倾斜 28 (+/-6) 度。该倾斜角与根据诱变和分子建模建议的受磷蛋白跨膜结构域模型计算出的倾斜角非常一致。磷酸化不会显着改变蛋白质的二级结构或方向。五聚体复合物被建模为由五个长螺旋（40 (+/-3) 个残基）组成的左手卷曲螺旋，从腔羧基末端跨膜延伸到细胞质中的磷酸化位点。螺旋束形成垂直的离子孔，其可能始于距膜表面一定距离（17 至 29 埃）处。基于上述，我们提出了一种受磷蛋白调节跨膜 Ca2+ 水平的机制，该机制考虑了其选择性离子电导和与 Ca2+ 泵的抑制关联。
Phospholamban is a 52 amino acid residue membrane protein involved with the regulation of calcium levels across sarcoplasmic reticulum membranes in cardiac muscle cells. The N-terminal 30 amino acid residues of the protein are largely hydrophilic and include two sites whose phosphorylation is thought to dissociate an inhibitory complex between phospholamban and Ca2+ ATPase. The C-terminal 22 amino acid residues are largely hydrophobic, anchor the protein in the membrane and are responsible for Ca2+ selective ion conductance. Specific interactions between the transmembrane domains stabilize a pentameric protein complex. We have obtained circular dichroism (CD), transmission Fourier transform infrared (FTIR) and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectra of the full-length protein and have compared these results to those from a 28 residue peptide that includes the transmembrane domain. Both proteins reconstituted into phospholipid membranes are largely alpha-helical by CD and FTIR. Polarized ATR-FTIR measurements show that both the cytosolic and transmembrane helices are oriented perpendicular to the membrane plane with a tilt of 28 (+/-6)degrees with respect to the membrane normal. This tilt angle is in close agreement to that calculated from a model for the transmembrane domain of phospholamban suggested by mutagenesis and molecular modeling. Phosphorylation does not significantly change the secondary structure or orientation of the protein. The pentameric complex is modeled as a left-handed coiled-coil of five long helices (40 (+/-3) residues) that extended across the membrane from the lumenal carboxy terminus to the phosphorylation site in the cytoplasm. The helix bundle forms a perpendicular ion pore that may begin at a distance (17 to 29 Angstrom) from the membrane surface. Based on the above, we propose a mechanism by which phospholamban regulates Ca2+ levels across membranes that takes into account both its selective ion conductance and inhibitory association with the Ca2+ pump.