Molecular Dynamics Flexible Fitting Simulations Identify New Models of the Closed State of the Cystic Fibrosis Transmembrane Conductance Regulator Protein

Cystic
fibrosis (CF) is a lethal, genetic disease found in particular
in humans of European origin which is caused by mutations in the cystic
fibrosis transmembrane conductance regulator (CFTR) chloride channel.
The search for CF therapies acting by modulating the impaired function
of mutant CFTR will be greatly advanced by high resolution structures
of CFTR in different states. To date, two medium resolution electron
microscopy (EM) structures of CFTR are available (one of a distant
zebrafish (Danio rerio) CFTR ortholog
and one of human CFTR). The two models are nearly identical to one
another, and both correspond to the inward-facing, nucleotide binding
domains (NBDs) separated, closed state of the channel. In addition,
lower resolution structural data are available for human CFTR in an
alternative conformation which likely features associated NBDs and
thus geometrically resembles the conducting state of the channel.
Multiple homology models of human CFTR in multiple states have been
developed over the years, yet their correspondence to the existing
structural information is unexplored. In this work we use molecular
dynamics flexible fitting (MDFF) simulations to refine two previously
described CFTR models based on the available cryo-EM map of the human protein. This map was recorded in the absence of ATP
and consequently represents closed-state CFTR yet its features likely
correspond to an NBD associated conformation of the protein. Accordingly,
the resulting models feature dimerized NBDs yet with no membrane traversing
pore. Moreover, the open probability of the new models as deduced
from the MDFF trajectories is significantly lower than that deduced
from control MD trajectories initiated from the starting models. We
propose that the new models correspond to a CFTR conformation which
to date was largely unexplored yet is one that is relevant to the
gating cycle of the protein. In particular this conformation may participate
in rapid channel opening and closing through small allosteric movements
controlled by nucleotide binding and dissociation events. Analyzing
the resulting trajectories (and not only the final models as is usually
the case), we demonstrate that the refined models have good stereochemical
properties and are also in favorable agreement with multiple experimental
data. Moreover, despite different starting points, the final models
share many common features. Finally, we propose that the combination
of high resolution cryo-EM maps, which are currently emerging from
multiple laboratories, and MDFF simulations will be of value for the
development of yet more reliable CFTR models as well as for the identification
of binding sites for CFTR modulators.

囊性纤维化（Cystic fibrosis, CF）是一种致死性遗传性疾病，尤其高发于欧洲血统人群，其致病机制为囊性纤维化跨膜传导调节因子（cystic fibrosis transmembrane conductance regulator, CFTR）氯离子通道发生突变。
寻找通过调控突变型CFTR功能受损状态以治疗CF的疗法，有赖于获取不同构象下CFTR的高分辨率结构。截至目前，已有两株中等分辨率的CFTR电子显微镜（electron microscopy, EM）结构被解析：一株来自亲缘关系较远的斑马鱼（Danio rerio）CFTR同源蛋白，另一株则来自人类CFTR。
这两个结构模型几乎完全一致，均对应通道处于面向胞内、核苷酸结合结构域（nucleotide binding domains, NBDs）分离的关闭状态。
此外，已有低分辨率结构数据显示人类CFTR处于另一种构象，该构象的NBDs可能发生结合，因此在几何结构上类似通道的导电状态。
多年来，学界已构建出多种状态下人类CFTR的多套同源建模模型，但这些模型与现有结构信息的匹配程度尚未得到探究。
本研究利用分子动力学柔性拟合（molecular dynamics flexible fitting, MDFF）模拟，基于已获取的人类CFTR冷冻电镜（cryo-electron microscopy, cryo-EM）图谱，对此前报道的两株CFTR模型进行了优化。
该图谱是在缺乏ATP的条件下采集的，因此代表的是关闭状态的CFTR，但其结构特征大概率对应蛋白的NBD结合构象。据此，优化得到的模型呈现出二聚化的NBDs，但不存在跨膜孔道。
此外，通过MDFF模拟轨迹推导得到的新模型开放概率，显著低于从初始模型出发的对照分子动力学模拟轨迹所推导的开放概率。
我们提出，新模型对应的CFTR构象在目前尚未被充分探究，但却与蛋白的门控循环密切相关。具体而言，该构象可能通过核苷酸结合与解离事件调控的小型变构运动，参与通道的快速开闭过程。
通过分析模拟轨迹（而非常规研究所采用的仅分析最终模型），我们证明优化后的模型具备良好的立体化学性质，同时与多项实验数据吻合度较高。
此外，尽管初始模型的构建起点不同，最终得到的模型仍共享诸多共同特征。
最后，我们认为，当前多实验室陆续发布的高分辨率冷冻电镜图谱与MDFF模拟相结合，将有助于开发更为可靠的CFTR模型，同时也可用于鉴定CFTR调节剂的结合位点。