Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2.
Proteins. 2011 Jul;79(7):2233-46. doi: 10.1002/prot.23048. Epub 2011 May 10.
The human immunodeficiency virus type 1 (HIV-1) genome encodes 18 proteins and 2 peptides. Four of these proteins encode high-affinity calmodulin-binding sites for which direct interactions with calmodulin have already been described. In this study, the HIV-1 proteome is queried using an algorithm that predicts calmodulin-binding sites revealing seven new putative calmodulin-binding sites including residues 34-56 of the transactivator of transcription (Tat). Tat is a 101-residue intrinsically disordered RNA-binding protein that plays a central role in the regulation of HIV-1 replication. Interactions between a Tat peptide (residues 34-56), melittin, a well-characterized calmodulin-binding peptide, and calmodulin were examined by direct binding studies, mass spectrometry, and fluorescence. The Tat peptide binds to both calcium-saturated and apo-calmodulin with a low micromolar affinity. Conformational changes induced in the Tat peptide were determined by circular dichroism, and residues in calmodulin that interact with the peptide were identified by HSQC NMR spectroscopy. Multiple interactions between HIV-1 proteins and calmodulin, a highly promiscuous signal transduction hub protein, may be an important mechanism by which the virus controls cell physiology.
人类免疫缺陷病毒 1 型（HIV-1）基因组编码 18 种蛋白质和 2 种肽。其中 4 种蛋白质编码高亲和力钙调蛋白结合位点，已经描述了它们与钙调蛋白的直接相互作用。在这项研究中，使用一种算法对 HIV-1 蛋白质组进行了查询，该算法预测了钙调蛋白结合位点，揭示了包括转录激活剂（Tat）的 34-56 位残基在内的 7 个新的推定钙调蛋白结合位点。Tat 是一种 101 个残基的固有无序 RNA 结合蛋白，在 HIV-1 复制的调控中发挥核心作用。通过直接结合研究、质谱和荧光，研究了 Tat 肽（残基 34-56）、蜂毒素（一种经过充分研究的钙调蛋白结合肽）和钙调蛋白之间的相互作用。Tat 肽与钙饱和和去垢蛋白结合的亲和力为低微摩尔。通过圆二色性测定了 Tat 肽的构象变化，并通过 HSQC NMR 光谱鉴定了与肽相互作用的钙调蛋白残基。HIV-1 蛋白与钙调蛋白的多种相互作用，钙调蛋白是一种高度混杂的信号转导枢纽蛋白，可能是病毒控制细胞生理学的重要机制。
