Kazazić Saša, Bertoša Branimir, Luić Marija, Mikleušević Goran, Tarnowski Krzysztof, Dadlez Michal, Narczyk Marta, Bzowska Agnieszka
Division of Physical Chemistry, Ruđer Bošković Institute, Zagreb, Croatia.
Division of Physical Chemistry, Faculty of Science at University of Zagreb, Zagreb, Croatia.
J Am Soc Mass Spectrom. 2016 Jan;27(1):73-82. doi: 10.1007/s13361-015-1239-2. Epub 2015 Sep 3.
The biologically active form of purine nucleoside phosphorylase (PNP) from Escherichia coli (EC 2.4.2.1) is a homohexamer unit, assembled as a trimer of dimers. Upon binding of phosphate, neighboring monomers adopt different active site conformations, described as open and closed. To get insight into the functions of the two distinctive active site conformations, virtually inactive Arg24Ala mutant is complexed with phosphate; all active sites are found to be in the open conformation. To understand how the sites of neighboring monomers communicate with each other, we have combined H/D exchange (H/DX) experiments with molecular dynamics (MD) simulations. Both methods point to the mobility of the enzyme, associated with a few flexible regions situated at the surface and within the dimer interface. Although H/DX provides an average extent of deuterium uptake for all six hexamer active sites, it was able to indicate the dynamic mechanism of cross-talk between monomers, allostery. Using this technique, it was found that phosphate binding to the wild type (WT) causes arrest of the molecular motion in backbone fragments that are flexible in a ligand-free state. This was not the case for the Arg24Ala mutant. Upon nucleoside substrate/inhibitor binding, some release of the phosphate-induced arrest is observed for the WT, whereas the opposite effects occur for the Arg24Ala mutant. MD simulations confirmed that phosphate is bound tightly in the closed active sites of the WT; conversely, in the open conformation of the active site of the WT phosphate is bound loosely moving towards the exit of the active site. In Arg24Ala mutant binary complex Pi is bound loosely, too.
来自大肠杆菌（EC 2.4.2.1）的嘌呤核苷磷酸化酶（PNP）的生物活性形式是一个同型六聚体单元，由二聚体三聚体组装而成。在结合磷酸盐后，相邻的单体采用不同的活性位点构象，分别描述为开放型和封闭型。为了深入了解这两种独特活性位点构象的功能，将几乎无活性的Arg24Ala突变体与磷酸盐复合；发现所有活性位点均处于开放构象。为了理解相邻单体的位点如何相互通信，我们将氢/氘交换（H/DX）实验与分子动力学（MD）模拟相结合。这两种方法都表明了该酶的流动性，与位于表面和二聚体界面内的几个柔性区域相关。尽管H/DX提供了所有六个六聚体活性位点的平均氘摄取程度，但它能够表明单体之间串扰、变构的动态机制。使用该技术发现，磷酸盐与野生型（WT）结合会导致在无配体状态下具有柔性的主链片段中的分子运动停滞。对于Arg24Ala突变体则并非如此。在核苷底物/抑制剂结合后，观察到WT的磷酸盐诱导的停滞有一些释放，而Arg24Ala突变体则出现相反的效果。MD模拟证实磷酸盐在WT的封闭活性位点中紧密结合；相反，在WT活性位点的开放构象中，磷酸盐结合松散，朝着活性位点的出口移动。在Arg24Ala突变体二元复合物中，磷酸根离子也结合松散。
