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首页> 外文期刊>Biochimica et biophysica acta: BBA: International journal of biochemistry, biophysics and molecular biololgy. Proteins and Proteomics >Stepwise construction of triple-helical heparin binding sites using peptide models
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Stepwise construction of triple-helical heparin binding sites using peptide models

机译：使用肽模型逐步构建三螺旋肝素结合位点

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摘要

The specific localization of the asymmetric form of acetylcholinesterase (AChE) in neuromuscular junctions results from the interaction of its collagen-like tail with heparan sulfate proteoglycans in the synaptic basal lamina. This interaction involves two heparin binding consensus sequences of the form XBBXB, where B is a basic residue, located in the triple-helical collagen tail: GRKGR for the N-terminal site and GKRGK for the C-terminal site. To explore the basis of the higher heparin affinity seen for the C-terminal site vs. the N-terminal site, two homologous series of (Gly-Xaa-Yaa)8 peptides were constructed to model these triple-helical binding sites. Individual tripeptide units from each heparin binding site were introduced in a stepwise fashion into a Gly-Pro-Hyp framework, until the consensus sequence and its surrounding triplets were recreated. As each additional triplet from the binding site is inserted to replace a host Gly-Pro-Hyp triplet, the triple-helix stability decreases, and the drop in thermal stability is close to that expected if each Gly-X-Y triplet contributed independently to global stability. CD spectroscopy and calorimetry show the stability of these AChE model peptides is increased by addition of heparin, confirming binding to heparin, and the lack of significant enthalpy change indicates the binding is largely electrostatic in nature. Displacement assays measure the strength of the peptide–heparin interaction, and indicate an inverse correlation between the peptide ability to bind heparin and its thermal stability. The model peptides for the C-terminal binding site show a greater heparin affinity than the peptide models for the N-terminal binding site only when residues surrounding the consensus sequence are included.
机译：乙酰胆碱酯酶（AChE）不对称形式在神经肌肉接头中的特定定位是由于其胶原样尾巴与突触基底膜中硫酸乙酰肝素蛋白聚糖的相互作用而引起的。这种相互作用涉及形式为XBBXB的两个肝素结合共有序列，其中B是位于三螺旋胶原尾部的基本残基：N末端位点为GRKGR，C末端位点为GKRGK。为了探索对于C末端位点相对于N末端位点而言更高的肝素亲和力的基础，构建了两个同源系列的（Gly-Xaa-Yaa）8肽来模拟这些三螺旋结合位点。将来自每个肝素结合位点的单个三肽单元逐步引入Gly-Pro-Hyp框架，直到重建共有序列及其周围的三联体。当插入来自结合位点的每个其他三联体以替换宿主Gly-Pro-Hyp三联体时，三螺旋的稳定性降低，并且如果每个Gly-XY三联体独立地对整体稳定性有所贡献，则热稳定性的下降接近预期的下降。 CD光谱法和量热法表明，通过添加肝素可增强这些AChE模型肽的稳定性，从而证实与肝素的结合，而缺乏明显的焓变表明该结合在本质上主要是静电的。位移测定法可测量肽与肝素相互作用的强度，并表明肽结合肝素的能力与其热稳定性之间呈负相关。仅当包含共有序列周围的残基时，用于C末端结合位点的模型肽才显示出比用于N末端结合位点的肽模型更大的肝素亲和力。

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