Energetics of outer membrane phospholipase A (OMPLA) dimerization.
Energetics of outer membrane phospholipase A (OMPLA) dimerization.
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DOI：
10.1016/j.jmb.2006.01.033
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发表时间：
2006-04
影响因子：
5.6
通讯作者：
A. Stanley;P. Chuawong;T. Hendrickson;K. Fleming
中科院分区：
文献类型：
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作者：
A. Stanley;P. Chuawong;T. Hendrickson;K. Fleming
外膜磷脂酶A（OMPLA）是一种广泛存在于革兰氏阴性菌中的保守的跨膜酶，与多种病原微生物的毒力有关。尽管存在许多高分辨率结构，但对蛋白质磷脂酶活性的调节还不清楚。先前的生物化学研究已经证明，OMPLA的二聚化是其磷脂酶活性的先决条件，并且已经在体外显示这种二聚化依赖于钙和底物结合。因此，为了充分理解OMPLA的调节，有必要了解蛋白质二聚体的稳定性及其受其效应分子影响的程度。我们用沉降平衡分析超离心法研究了洗涤剂胶束中大肠杆菌OMPLA二聚化的能量学。我们发现，钙对二聚体的稳定性贡献相对较小，而与底物酰基链的相互作用是稳定酶的二聚体构象的主要力量。由此产生的热力学循环表明效应分子之间的相互作用是加性的。这些能量测量不仅提供了对OMPLA激活的深入了解，而且它们还代表了对跨膜β-桶的缔合能量学的首次定量研究。这一热力学研究使我们能够开始解决蛋白质-蛋白质界面之间的差异，在跨膜蛋白与螺旋折叠的β-桶折叠，并更充分地了解参与膜蛋白相互作用的力量。
Outer membrane phospholipase A (OMPLA) is a widely conserved transmembrane enzyme found in Gram-negative bacteria, and it is implicated in the virulence of a number of pathogenic organisms. The regulation of the protein's phospholipase activity is not well understood despite the existence of a number of high resolution structures. Previous biochemical studies have demonstrated that dimerization of OMPLA is a prerequisite for its phospholipase activity, and it has been shown in vitro that this dimerization is dependent on calcium and substrate binding. Therefore, to fully understand the regulation of OMPLA, it is necessary to understand the stability of the protein dimer and the extent to which it is influenced by its effector molecules. We have used sedimentation equilibrium analytical ultracentrifugation to dissect the energetics of Escherichia coli OMPLA dimerization in detergent micelles. We find that calcium contributes relatively little stability to the dimer, while interactions with the substrate acyl chain are the predominant force in stabilizing the dimeric conformation of the enzyme. The resulting thermodynamic cycle suggests that interactions between effector molecules are additive. These energetic measurements not only provide insight into the activation of OMPLA, but they also represent the first quantitative investigation of the association energetics of a transmembrane β-barrel. This thermodynamic study allows us to begin to address the differences between protein–protein interfaces in transmembrane proteins with a helical fold to those of a β-barrel fold and to more fully understand the forces involved in membrane protein interactions.