BACKGROUND 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. RESULTS 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. CONCLUSIONS 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.

中文翻译：

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Rosetta Broker用于膜蛋白结构预测：浓缩核苷转运蛋白3和促肾上腺皮质激素释放因子受体1测试案例。

背景技术由于沉积在蛋白质数据库中的结构数据有限，膜蛋白难以用于结构预测。膜蛋白结构预测的大多数计算方法都基于比较模型。只有很少的针对该独特蛋白质家族的从头方法。在这项工作中，使用了这种从头方法的例子来从结构和功能上表征溶质载体转运蛋白和G蛋白偶联受体的不同膜蛋白家族的两个代表。在两个测试用例中使用了著名的Rosetta程序及其名为Broker的协议之一。第一种情况是人类集中核苷转运蛋白3（hCNT3）同型三聚体的三个N端跨膜螺旋的从头结构预测，属于溶质载体28家族转运蛋白（SLC28）。第二种情况涉及属于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的大规模提纯。结果使用hCNT3同源三聚体的向内模型来提出其单核苷酸多态性的功能影响。此外，未配体的hCNT3模型的100 ns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迁移性。CRFR1同源模型的跨膜螺旋的大规模完善导致其相对于CRFR1的晶体结构，特别是在结合位点区域的准确性的显着提高。因此，拮抗剂CP-376395可与Autodock VINA对接至CRFR1模型，而不会发生任何空间碰撞。结论提出的工作表明Rosetta Broker可以作为解决涉及蛋白质生物学的各种问题的多功能工具。这里介绍的从头膜蛋白质结构预测的两个不同示例为蛋白质生物学的三个主要领域提供了重要见解。即，向内的hCNT3同型三聚体系统的动力学，拮抗剂结合后CRFR1受体的结构变化，以及最后两者中单核苷酸多态性的作用，