Rosenlöw Joakim, Isaksson Linnéa, Mayzel Maxim, Lengqvist Johan, Orekhov Vladislav Y
The Swedish NMR Centre, University of Gothenburg, Gothenburg, Sweden.
Proteomics Core Facility at Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
PLoS One. 2014 Apr 25;9(4):e96199. doi: 10.1371/journal.pone.0096199. eCollection 2014.
Intrinsically disordered proteins are found extensively in cell signaling pathways where they often are targets of posttranslational modifications e.g. phosphorylation. Such modifications can sometimes induce or disrupt secondary structure elements present in the modified protein. CD79a and CD79b are membrane-spanning, signal-transducing components of the B-cell receptor. The cytosolic domains of these proteins are intrinsically disordered and each has an immunoreceptor tyrosine-based activation motif (ITAM). When an antigen binds to the receptor, conserved tyrosines located in the ITAMs are phosphorylated which initiate further downstream signaling. Here we use NMR spectroscopy to examine the secondary structure propensity of the cytosolic domains of CD79a and CD79b in vitro before and after phosphorylation. The phosphorylation patterns are identified through analysis of changes of backbone chemical shifts found for the affected tyrosines and neighboring residues. The number of the phosphorylated sites is confirmed by mass spectrometry. The secondary structure propensities are calculated using the method of intrinsic referencing, where the reference random coil chemical shifts are measured for the same protein under denaturing conditions. Our analysis revealed that CD79a and CD79b both have an overall propensity for α-helical structure that is greatest in the C-terminal region of the ITAM. Phosphorylation of CD79a caused a decrease in helical propensity in the C-terminal ITAM region. For CD79b, the opposite was observed and phosphorylation resulted in an increase of helical propensity in the C-terminal part.
内在无序蛋白广泛存在于细胞信号通路中，它们常常是翻译后修饰（例如磷酸化）的靶点。此类修饰有时会诱导或破坏被修饰蛋白中存在的二级结构元件。CD79a和CD79b是B细胞受体的跨膜信号转导成分。这些蛋白的胞质结构域是内在无序的，且每个都有一个基于免疫受体酪氨酸的激活基序（ITAM）。当抗原与受体结合时，位于ITAM中的保守酪氨酸会被磷酸化，从而启动进一步的下游信号传导。在这里，我们使用核磁共振光谱法来检测CD79a和CD79b的胞质结构域在体外磷酸化前后的二级结构倾向。通过分析受影响酪氨酸及其相邻残基的主链化学位移变化来确定磷酸化模式。磷酸化位点的数量通过质谱法确认。使用内在参照法计算二级结构倾向，即在变性条件下对同一蛋白测量参照无规卷曲化学位移。我们的分析表明，CD79a和CD79b在ITAM的C端区域都总体上倾向于形成α螺旋结构，且这种倾向最为明显。CD79a的磷酸化导致C端ITAM区域的螺旋倾向降低。对于CD79b，则观察到相反的情况，磷酸化导致C端部分的螺旋倾向增加。
