Experimental phasing with vanadium and application to nucleotide-binding membrane proteins.
Experimental phasing with vanadium and application to nucleotide-binding membrane proteins.
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DOI：
10.1107/s2052252520012312
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发表时间：
2020-11-01
期刊：
影响因子：
3.9
通讯作者：
Wagner A
中科院分区：
文献类型：
--
作者：
El Omari K;Mohamad N;Bountra K;Duman R;Romano M;Schlegel K;Kwong HS;Mykhaylyk V;Olesen C;Moller JV;Bublitz M;Beis K;Wagner A
膜蛋白的晶体结构测定可能具有挑战性。在此，描述了使用钒定相作为一种替代性的实验定相方法。
可溶性蛋白和膜蛋白的结构测定可能会受到晶体学相位问题的阻碍，尤其是在没有合适的同源结构的情况下。实验定相是新结构的首选方法；然而，它通常需要重原子衍生化，这可能既困难又耗时。在此，报道了一种通过钒定相来获取蛋白质结构测定的实验相位的新颖且快速的方法。钒酸盐是磷酸转移反应的过渡态模拟物，它具有特异性结合众多催化该反应的酶的活性位点的优势。通过测定三种不同的蛋白质 - 钒复合物的结构，验证了钒定相的适用性，其中两种是整合膜蛋白：兔肌浆网Ca²⁺ - ATP酶、来自大肠杆菌的抗菌肽ATP结合盒转运体McjD以及来自牛的可溶性酶核糖核酸酶A。即使在低分辨率下，且尽管数据存在严重的各向异性，钒定相也取得了成功。这种方法原则上适用于大量的蛋白质，涵盖了酶学委员会分类的七类中的六类。它完全依赖于蛋白质的特定化学性质，不需要任何修饰，使其成为定相工具包中一个非常强大的补充。除了这种技术的定相能力外，蛋白质 - 钒复合物还为所研究蛋白质的反应机制提供了详细的见解。
Crystal structure determination of membrane proteins can be challenging. Here, the use of vanadium phasing as an alternative experimental phasing method is described. The structure determination of soluble and membrane proteins can be hindered by the crystallographic phase problem, especially in the absence of a suitable homologous structure. Experimental phasing is the method of choice for novel structures; however, it often requires heavy-atom derivatization, which can be difficult and time-consuming. Here, a novel and rapid method to obtain experimental phases for protein structure determination by vanadium phasing is reported. Vanadate is a transition-state mimic of phosphoryl-transfer reactions and it has the advantage of binding specifically to the active site of numerous enzymes catalyzing this reaction. The applicability of vanadium phasing has been validated by determining the structures of three different protein–vanadium complexes, two of which are integral membrane proteins: the rabbit sarcoplasmic reticulum Ca2+-ATPase, the antibacterial peptide ATP-binding cassette transporter McjD from Escherichia coli and the soluble enzyme RNAse A from Bos taurus. Vanadium phasing was successful even at low resolution and despite severe anisotropy in the data. This method is principally applicable to a large number of proteins, representing six of the seven Enzyme Commission classes. It relies exclusively on the specific chemistry of the protein and it does not require any modifications, making it a very powerful addition to the phasing toolkit. In addition to the phasing power of this technique, the protein–vanadium complexes also provide detailed insights into the reaction mechanisms of the studied proteins.