Myllykoski Matti, Baumann Anne, Hensley Kenneth, Kursula Petri
Faculty of Biochemistry and Molecular Medicine and Biocenter Oulu, University of Oulu, Oulu, Finland.
Department of Biomedicine, University of Bergen, Bergen, Norway.
Amino Acids. 2017 Apr;49(4):747-759. doi: 10.1007/s00726-016-2376-z. Epub 2017 Jan 2.
Collapsin response mediator protein 2 (CRMP-2) is a neuronal protein involved in axonal pathfinding. Intense research is focusing on its role in various neurological diseases. Despite a wealth of studies, not much is known about the molecular mechanisms of CRMP-2 function in vivo. The detailed structure-function relationships of CRMP-2 have also largely remained unknown, in part due to the fact that the available crystal structures lack the C-terminal tail, which is known to be a target for many post-translational modifications and protein interactions. Although CRMP-2, and other CRMPs, belong to the dihydropyrimidinase family, they have lost the enzymatic active site. Drug candidates for CRMP-2-related processes have come up during the recent years, but no reports of CRMP-2 complexes with small molecules have emerged. Here, CRMP-2 was studied at 1.25-Å resolution using X-ray crystallography. In addition, ligands were docked into the homotetrameric structure, and the C-terminal tail of CRMP-2 was produced recombinantly and analyzed. We have obtained the human CRMP-2 crystal structure at atomic resolution and could identify small-molecule binding pockets in the protein. Structures obtained in different crystal forms highlight flexible regions near possible ligand-binding pockets. We also used the CRMP-2 structure to analyze known or suggested post-translational modifications at the 3D structural level. The high-resolution CRMP-2 structure was also used for docking experiments with the sulfur amino acid metabolite lanthionine ketimine and its ester. We show that the C-terminal tail is intrinsically disordered, but it has conserved segments that may act as interaction sites. Our data provide the most accurate structural data on CRMPs to date and will be useful in further computational and experimental studies on CRMP-2, its function, and its binding to small-molecule ligands.
塌陷反应介导蛋白2（CRMP-2）是一种参与轴突导向的神经元蛋白。大量研究聚焦于其在各种神经疾病中的作用。尽管已有大量研究，但关于CRMP-2在体内功能的分子机制仍知之甚少。CRMP-2详细的结构-功能关系在很大程度上也尚不明确，部分原因是现有的晶体结构缺少C末端尾巴，而C末端尾巴已知是许多翻译后修饰和蛋白质相互作用的靶点。尽管CRMP-2以及其他CRMPs属于二氢嘧啶酶家族，但它们已失去酶活性位点。近年来出现了与CRMP-2相关过程的候选药物，但尚未有CRMP-2与小分子复合物的报道。在此，利用X射线晶体学以1.25埃的分辨率对CRMP-2进行了研究。此外，将配体对接至同四聚体结构中，并重组产生了CRMP-2的C末端尾巴并进行分析。我们获得了原子分辨率的人CRMP-2晶体结构，并能够识别该蛋白中的小分子结合口袋。不同晶体形式获得的结构突出了可能的配体结合口袋附近的柔性区域。我们还利用CRMP-2结构在三维结构水平上分析已知或推测的翻译后修饰。高分辨率的CRMP-2结构还用于与含硫氨基酸代谢物羊毛硫氨酸酮亚胺及其酯的对接实验。我们表明C末端尾巴本质上是无序的，但具有可能作为相互作用位点的保守片段。我们的数据提供了迄今为止关于CRMPs最准确的结构数据，将有助于对CRMP-2及其功能以及与小分子配体结合的进一步计算和实验研究。
