Pierced Lasso Topology Controls Function in Leptin
Pierced Lasso Topology Controls Function in Leptin
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刺穿套索拓扑控制瘦素的功能
DOI：
10.1021/acs.jpcb.6b11506
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发表时间：
2017
期刊：
影响因子：
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通讯作者：
Onuchic, José Nelson
中科院分区：
文献类型：
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作者：
Haglund, Ellinor;Pilko, Anna;Wollman, Roy;Jennings, Patricia Ann;Onuchic, José Nelson
蛋白质工程在药物设计和治疗中是一个强有力的工具，在药物设计和治疗中，通常引入二硫键来稳定蛋白质。然而，这些键也引入了经常被忽视的共价环。这些环可能会将蛋白质主干缠绕在相反的两侧，导致一种“打结”的拓扑结构，形成所谓的穿孔套索(PL)。在这个优雅的系统中，“结”通过一个单独的二硫键连接在一起，其中部分多肽链被穿过。这些共价环的大小和位置可以通过体外蛋白质设计来操纵，而大自然则利用多态性来切换PL拓扑。PL蛋白Leptin显示了N末端残基的遗传修饰，在相同的序列中添加了第三个半胱氨酸。为了了解这些不同拓扑的线程机制，我们设计了三个循环变体来模拟多态序列。这为正在研究的系统增加了优雅，因为它允许产生三个可能的共价环；它们是原始的野生型C-末端环蛋白，完全循环的无线蛋白和N-末端环蛋白，负责不同的套索拓扑。线圈的尺寸随着线圈尺寸的增加，将穿线机构从打滑机构改变为封堵机构。有趣的是，天然蛋白质结构的基态在很大程度上没有受到影响，但生物分析表明，通过适当控制线状状态下的动力学，活性可以最大化。具有适当构象动力学的线状拓扑结构对于受体的相互作用和体内信号通路的激活是重要的。
Protein engineering is a powerful tool in drug design and therapeutics, where disulphide bridges are commonly introduced to stabilize proteins. However, these bonds also introduce covalent loops, which are often neglected. These loops may entrap the protein backbone on opposite sides, leading to a “knotted” topology, forming a so-called Pierced Lasso (PL). In this elegant system, the “knot” is held together with a single disulphide bridge where part of the polypeptide chain is threaded through. The size and position of these covalent loops can be manipulated through protein design in vitro, whereas nature uses polymorphism to switch the PL topology. The PL protein leptin shows genetic modification of an N-terminal residue, adding a third cysteine to the same sequence. In an effort to understand the mechanism of threading of these diverse topologies, we designed three loop variants to mimic the polymorphic sequence. This adds elegance to the system under study, as it allows the generation of three possible covalent loops; they are the original wild-type C-terminal loop protein, the fully circularized unthreaded protein, and the N-terminal loop protein, responsible for different lasso topologies. The size of the loop changes the threading mechanism from a slipknotting to a plugging mechanism, with increasing loop size. Interestingly, the ground state of the native protein structure is largely unaffected, but biological assays show that the activity is maximized by properly controlled dynamics in the threaded state. A threaded topology with proper conformational dynamics is important for receptor interaction and activation of the signaling pathways in vivo.