Latek Dorota
Faculty of Chemistry, University of Warsaw, Pasteur St. 1, 02-093, Warsaw, Poland.
BMC Struct Biol. 2017 Aug 3;17(1):8. doi: 10.1186/s12900-017-0078-8.
Membrane proteins are difficult targets for structure prediction due to the limited structural data deposited in Protein Data Bank. Most computational methods for membrane protein structure prediction are based on the comparative modeling. There are only few de novo methods targeting that distinct protein family. In this work an example of such de novo method was used to structurally and functionally characterize two representatives of distinct membrane proteins families of solute carrier transporters and G protein-coupled receptors. The well-known Rosetta program and one of its protocols named Broker was used in two test cases. The first case was de novo structure prediction of three N-terminal transmembrane helices of the human concentrative nucleoside transporter 3 (hCNT3) homotrimer belonging to the solute carrier 28 family of transporters (SLC28). The second case concerned the large scale refinement of transmembrane helices of a homology model of the corticotropin-releasing factor receptor 1 (CRFR1) belonging to the G protein-coupled receptors family.
The inward-facing model of the hCNT3 homotrimer was used to propose the functional impact of its single nucleotide polymorphisms. Additionally, the 100 ns molecular dynamics simulation of the unliganded hCNT3 model confirmed its validity and revealed mobility of the selected binding site and homotrimer interface residues. The large scale refinement of transmembrane helices of the CRFR1 homology model resulted in the significant improvement of its accuracy with respect to the crystal structure of CRFR1, especially in the binding site area. Consequently, the antagonist CP-376395 could be docked with Autodock VINA to the CRFR1 model without any steric clashes.
The presented work demonstrated that Rosetta Broker can be a versatile tool for solving various issues referring to protein biology. Two distinct examples of de novo membrane protein structure prediction presented here provided important insights into three major areas of protein biology. Namely, the dynamics of the inward-facing hCNT3 homotrimer system, the structural changes of the CRFR1 receptor upon the antagonist binding and finally, the role of single nucleotide polymorphisms in both, hCNT3 and CRFR1 proteins, were investigated.
由于蛋白质数据库中存储的结构数据有限，膜蛋白是结构预测的困难靶点。大多数膜蛋白结构预测的计算方法基于比较建模。针对该独特蛋白家族的从头预测方法很少。在这项工作中，使用了一种此类从头预测方法的示例，对溶质载体转运蛋白和G蛋白偶联受体这两个不同膜蛋白家族的代表进行结构和功能表征。在两个测试案例中使用了著名的Rosetta程序及其名为Broker的一个协议。第一个案例是对属于溶质载体28转运蛋白家族（SLC28）的人浓缩核苷转运蛋白3（hCNT3）同三聚体的三个N端跨膜螺旋进行从头结构预测。第二个案例涉及对属于G蛋白偶联受体家族的促肾上腺皮质激素释放因子受体1（CRFR1）同源模型的跨膜螺旋进行大规模优化。
hCNT3同三聚体的内向型模型被用于推测其单核苷酸多态性的功能影响。此外，未结合配体的hCNT3模型的100纳秒分子动力学模拟证实了其有效性，并揭示了所选结合位点和同三聚体界面残基的流动性。CRFR1同源模型跨膜螺旋的大规模优化导致其相对于CRFR1晶体结构的准确性有显著提高，特别是在结合位点区域。因此，拮抗剂CP - 376395可以使用Autodock VINA对接至CRFR1模型，且没有任何空间冲突。
本研究表明Rosetta Broker可以成为解决蛋白质生物学各种问题的通用工具。这里展示的两个不同的膜蛋白从头结构预测示例为蛋白质生物学的三个主要领域提供了重要见解。即，研究了内向型hCNT3同三聚体系统的动力学、拮抗剂结合后CRFR1受体的结构变化，以及最后hCNT3和CRFR1蛋白中单个核苷酸多态性的作用。
