Department of Physical Chemistry and Institute of Biotechnology, Faculty of Sciences, University of Granada, Granada, Spain.
PLoS One. 2014 Feb 26;9(2):e90030. doi: 10.1371/journal.pone.0090030. eCollection 2014.
The relative promiscuity of hub proteins such as postsynaptic density protein-95 (PSD-95) can be achieved by alternative splicing, allosteric regulation, and post-translational modifications, the latter of which is the most efficient method of accelerating cellular responses to environmental changes in vivo. Here, a mutational approach was used to determine the impact of phosphorylation and succinimidation post-translational modifications on the binding affinity of the postsynaptic density protein-95/discs large/zonula occludens-1 (PDZ3) domain of PSD-95. Molecular dynamics simulations revealed that the binding affinity of this domain is influenced by an interplay between salt-bridges linking the α3 helix, the β2-β3 loop and the positively charged Lys residues in its high-affinity hexapeptide ligand KKETAV. The α3 helix is an extra structural element that is not present in other PDZ domains, which links PDZ3 with the following SH3 domain in the PSD-95 protein. This regulatory mechanism was confirmed experimentally via thermodynamic and NMR chemical shift perturbation analyses, discarding intra-domain long-range effects. Taken together, the results presented here reveal the molecular basis of the regulatory role of the α3 extra-element and the effects of post-translational modifications of PDZ3 on its binding affinity, both energetically and dynamically.
诸如突触后致密蛋白95（PSD-95）等枢纽蛋白的相对混杂性可通过可变剪接、变构调节和翻译后修饰来实现，其中翻译后修饰是体内加速细胞对环境变化做出反应的最有效方法。在此，采用突变方法来确定磷酸化和琥珀酰亚胺化翻译后修饰对PSD-95的突触后致密蛋白95/盘状大蛋白/紧密连接蛋白1（PDZ3）结构域结合亲和力的影响。分子动力学模拟表明，该结构域的结合亲和力受连接α3螺旋、β2-β3环与其高亲和力六肽配体KKETAV中带正电荷的赖氨酸残基的盐桥之间相互作用的影响。α3螺旋是一个额外的结构元件，不存在于其他PDZ结构域中，它将PSD-95蛋白中的PDZ3与下游的SH3结构域相连。通过热力学和核磁共振化学位移扰动分析实验证实了这种调节机制，排除了结构域内的长程效应。综上所述，本文给出的结果揭示了α3额外元件的调节作用以及PDZ3的翻译后修饰对其结合亲和力在能量和动力学方面的影响的分子基础。
