Mehler Ernest L, Hassan Sergio A, Kortagere Sandhya, Weinstein Harel
Department of Physiology and Biophysics, Weill Medical College of Cornell University, New York, New York 10021, USA.
Proteins. 2006 Aug 15;64(3):673-90. doi: 10.1002/prot.21022.
With the help of the crystal structure of rhodopsin an ab initio method has been developed to calculate the three-dimensional structure of the loops that connect the transmembrane helices (TMHs). The goal of this procedure is to calculate the loop structures in other G-protein coupled receptors (GPCRs) for which only model coordinates of the TMHs are available. To mimic this situation a construct of rhodopsin was used that only includes the experimental coordinates of the TMHs while the rest of the structure, including the terminal domains, has been removed. To calculate the structure of the loops a method was designed based on Monte Carlo (MC) simulations which use a temperature annealing protocol, and a scaled collective variables (SCV) technique with proper structural constraints. Because only part of the protein is used in the calculations the usual approach of modeling loops, which consists of finding a single, lowest energy conformation of the system, is abandoned because such a single structure may not be a representative member of the native ensemble. Instead, the method was designed to generate structural ensembles from which the single lowest free energy ensemble is identified as representative of the native folding of the loop. To find the native ensemble a successive series of SCV-MC simulations are carried out to allow the loops to undergo structural changes in a controlled manner. To increase the chances of finding the native funnel for the loop, some of the SCV-MC simulations are carried out at elevated temperatures. The native ensemble can be identified by an MC search starting from any conformation already in the native funnel. The hypothesis is that native structures are trapped in the conformational space because of the high-energy barriers that surround the native funnel. The existence of such ensembles is demonstrated by generating multiple copies of the loops from their crystal structures in rhodopsin and carrying out an extended SCV-MC search. For the extracellular loops e1 and e3, and the intracellular loop i1 that were used in this work, the procedure resulted in dense clusters of structures with Calpha-RMSD approximately 0.5 angstroms. To test the predictive power of the method the crystal structure of each loop was replaced by its extended conformations. For e1 and i1 the procedure identifies native clusters with Calpha-RMSD approximately 0.5 angstroms and good structural overlap of the side chains; for e3, two clusters were found with Calpha-RMSD approximately 1.1 angstroms each, but with poor overlap of the side chains. Further searching led to a single cluster with lower Calpha-RMSD but higher energy than the two previous clusters. This discrepancy was found to be due to the missing elements in the constructs available from experiment for use in the calculations. Because this problem will likely appear whenever parts of the structural information are missing, possible solutions are discussed.
借助视紫红质的晶体结构，已开发出一种从头算方法来计算连接跨膜螺旋（TMHs）的环的三维结构。此程序的目标是计算其他G蛋白偶联受体（GPCRs）中的环结构，对于这些受体，仅可获得TMHs的模型坐标。为模拟这种情况，使用了一种视紫红质构建体，其仅包含TMHs的实验坐标，而结构的其余部分，包括末端结构域，已被去除。为计算环的结构，设计了一种基于蒙特卡罗（MC）模拟的方法，该方法使用温度退火协议以及具有适当结构约束的缩放集体变量（SCV）技术。由于在计算中仅使用了蛋白质的一部分，因此放弃了通常的环建模方法，即寻找系统的单一最低能量构象，因为这样的单一结构可能不是天然系综的代表性成员。相反，该方法旨在生成结构系综，从中将单一最低自由能系综识别为环的天然折叠的代表。为找到天然系综，进行了一系列连续的SCV-MC模拟，以使环以可控方式发生结构变化。为增加找到环的天然漏斗的机会，一些SCV-MC模拟在升高的温度下进行。天然系综可通过从天然漏斗中已有的任何构象开始的MC搜索来识别。假设是天然结构被困在构象空间中是因为围绕天然漏斗的高能障碍。通过从视紫红质的晶体结构生成环的多个副本并进行扩展的SCV-MC搜索，证明了这种系综的存在。对于本工作中使用的细胞外环e1和e3以及细胞内环i1，该程序产生了结构密集簇，其Cα-RMSD约为0.5埃。为测试该方法的预测能力，将每个环的晶体结构替换为其扩展构象。对于e1和i1，该程序识别出Cα-RMSD约为0.5埃且侧链具有良好结构重叠的天然簇；对于e3，发现了两个Cα-RMSD约为1.1埃的簇，但侧链重叠较差。进一步搜索得到了一个Cα-RMSD较低但能量高于前两个簇的单一簇。发现这种差异是由于计算中可用的实验构建体中缺少元素。由于每当部分结构信息缺失时可能都会出现这个问题，因此讨论了可能的解决方案。
