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首页> 外文期刊>Journal of Molecular Biology >Analysis of the thermodynamics of binding of an SH3 domain to proline-rich peptides using a chimeric fusion protein
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Analysis of the thermodynamics of binding of an SH3 domain to proline-rich peptides using a chimeric fusion protein

机译：使用嵌合融合蛋白分析SH3域与富含脯氨酸的肽结合的热力学

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A complete understanding of the thermodynamic determinants of binding between SH3 domains and proline-rich peptides is crucial to the development of rational strategies for designing ligands for these important domains. Recently we engineered a single-chain chimeric protein by fusing the alpha-spectrin Src homology region 3 (SH3) domain to the decapeptide APSYSPPPPP (p41). This chimera mimics the structural and energetic features of the interaction between SH3 domains and proline-rich peptides. Here we show that analysing the unfolding thermodynamics of single-point mutants of this chimeric fusion protein constitutes a very useful approach to deciphering the thermodynamics of SH3-ligand interactions. To this end, we investigated the contribution of each proline residue of the ligand sequence to the SH3-peptide interaction by producing six single Pro-Ala mutants of the chimeric protein and analysing their unfolding thermodynamics by differential scanning calorimetry (DSC). Structural analyses of the mutant chimeras by circular dichroism, fluorescence and NMR together with NMR-relaxation measurements indicate conformational flexibility at the binding interface, which is strongly affected by the different Pro-Ala mutations. An analysis of the DSC thermograms on the basis of a three-state unfolding model has allowed us to distinguish and separate the thermodynamic magnitudes of the interaction at the binding interface. The model assumes equilibrium between the "unbound" and "bound" states at the SH3-peptide binding interface. The resulting thermodynamic magnitudes classify the different proline residues according to their importance in the interaction as P2 similar to P7 similar to P10>P9 similar to P6>P8, which agrees well with Lim's model for the interaction between SH3 domains and proline-rich peptides. In addition, the thermodynamic signature of the interaction is the same as that usually found for this type of binding, with a strong enthalpy-entropy compensation for all the mutants. This compensation appears to derive from an increase in conformational flexibility concomitant to the weakening of the interactions at the binding interface. We conclude that our approach, based on DSC and site-directed mutagenesis analysis of chimeric fusion proteins, may serve as a suitable tool to analyse the energetics of weak biomolecular interactions such as those involving SH3 domains. (c) 2007 Elsevier Ltd. All rights reserved.
机译：完整了解SH3域和富含脯氨酸的肽之间结合的热力学决定因素对于开发为这些重要域设计配体的合理策略至关重要。最近，我们通过将α-血影蛋白Src同源区域3（SH3）域融合到十肽APSYSPPPPP（p41）中，设计了单链嵌合蛋白。该嵌合体模仿SH3结构域和富含脯氨酸的肽之间相互作用的结构和能量特征。在这里我们表明，分析这种嵌合融合蛋白的单点突变体的展开热力学构成了破译SH3-配体相互作用的热力学的非常有用的方法。为此，我们通过产生嵌合蛋白的六个单个Pro-Ala突变体并通过差示扫描量热法（DSC）分析了它们的展开热力学，研究了配体序列中每个脯氨酸残基对SH3-肽相互作用的贡献。通过圆二色性，荧光和NMR以及NMR弛豫测量对突变体嵌合体进行结构分析表明，结合界面处的构象柔韧性受到不同Pro-Ala突变的强烈影响。在三态展开模型的基础上对DSC热谱图的分析使我们能够区分和分离结合界面处相互作用的热力学强度。该模型假定SH3-肽结合界面在“未结合”和“结合”状态之间达到平衡。产生的热力学强度根据不同的脯氨酸残基在相互作用中的重要性将其分类为P2相似，P7相似，P10相似，P9相似，P6相似，P8相似，这与Lim模型中SH3域与富含脯氨酸的肽之间的相互作用非常吻合。此外，相互作用的热力学特征与这种结合通常发现的特征相同，对所有突变体都有很强的焓-熵补偿。这种补偿似乎来自伴随结合界面相互作用减弱的构象柔性的增加。我们得出的结论是，基于DSC和嵌合融合蛋白的定点诱变分析，我们的方法可作为分析弱生物分子相互作用（例如涉及SH3结构域）的能量学的合适工具。 （c）2007 Elsevier Ltd.保留所有权利。

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