Haglund Ellinor, Pilko Anna, Wollman Roy, Jennings Patricia Ann, Onuchic José Nelson
Center for Theoretical Biological Physics (CTBP) and Departments of Physics and Astronomy, Chemistry and Biosciences, Rice University , Houston, Texas, United States.
Department of Chemistry and Biochemistry, The University of California, San Diego (UCSD) , La Jolla, California, United States.
J Phys Chem B. 2017 Feb 2;121(4):706-718. doi: 10.1021/acs.jpcb.6b11506. Epub 2017 Jan 18.
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.
蛋白质工程是药物设计和治疗领域的强大工具，在该领域中，二硫键常被引入以稳定蛋白质。然而，这些键也会引入共价环，而这些环常常被忽视。这些环可能会在相对的两侧困住蛋白质主链，导致形成“打结”的拓扑结构，即所谓的穿孔套索（PL）。在这个精妙的系统中，“结”由一个二硫键维系在一起，多肽链的一部分从中穿过。这些共价环的大小和位置可以通过体外蛋白质设计进行操控，而自然界则利用多态性来切换PL拓扑结构。PL蛋白瘦素显示出N端残基的基因修饰，在同一序列中添加了第三个半胱氨酸。为了理解这些不同拓扑结构的穿线机制，我们设计了三种环变体来模拟多态序列。这为正在研究的系统增添了精妙之处，因为它允许生成三种可能的共价环；它们分别是原始的野生型C端环蛋白、完全环化的无穿线蛋白以及负责不同套索拓扑结构的N端环蛋白。随着环大小的增加，环的大小会将穿线机制从活结机制转变为堵塞机制。有趣的是，天然蛋白质结构的基态在很大程度上不受影响，但生物学分析表明，在穿线状态下通过适当控制动力学可使活性最大化。具有适当构象动力学的穿线拓扑结构对于体内受体相互作用和信号通路的激活很重要。
