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首页> 外文期刊>Biochemistry >Structure and Dynamics of GeoCyp: A Thermophilic Cyclophilin with a Novel Substrate Binding Mechanism That Functions Efficiently at Low Temperatures
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Structure and Dynamics of GeoCyp: A Thermophilic Cyclophilin with a Novel Substrate Binding Mechanism That Functions Efficiently at Low Temperatures

机译：GeoCyp的结构和动力学：嗜热亲环蛋白，具有新型底物结合机制，可在低温下有效发挥作用

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Thermophilic proteins have found extensive use in research and industrial applications because of their high stability and functionality at elevated temperatures while simultaneously providing valuable insight into our understanding of protein folding, stability, dynamics, and function. Cyclophilins, constituting a ubiquitously expressed family of peptidyl prolyl isomerases with a range of biological functions and disease associations, have been utilized both for conferring stress tolerances and in exploring the link between conformational dynamics and enzymatic function. To date, however, no active thermophilic cyclophilin has been fully biophysically characterized. Here, we determine the structure of a thermophilic cyclophilin (GeoCyp) from Geobacillus kaustophilus, characterize its dynamic motions over several time scales using an array of methodologies that include chemical shift-based methods and relaxation experiments over a range of temperatures, and measure catalytic activity over a range of temperatures to compare its structure, dynamics, and function to those of a mesophilic counterpart, human cyclophilin A (CypA). Unlike those of most thermophile/mesophile pairs, GeoCyp catalysis is not substantially impaired at low temperatures as compared to that of CypA, retaining similar to 70% of the activity of its mesophilic counterpart. Examination of substrate-bound ensembles reveals a mechanism by which the two cyclophilins may have adapted to their environments through altering dynamic loop motions and a critical residue that acts as a clamp to regulate substrate binding differentially in CypA and GeoCyp. Fast time scale (pico- to nanosecond) dynamics are largely conserved between the two proteins, in accordance with the high degree of structural similarity, although differences do exist in their temperature dependencies. Slower (microsecond) time scale motions are likewise localized to similar regions in the two proteins with some variability in their magnitudes yet do not exhibit significant temperature dependencies in either enzyme.
机译：嗜热蛋白由于在高温下的高稳定性和功能性而在研究和工业应用中得到了广泛应用，同时为我们对蛋白折叠，稳定性，动力学和功能的理解提供了宝贵的见识。亲环蛋白构成了普遍表达的具有一系列生物学功能和疾病关联的肽基脯氨酰异构酶家族，已经被用于赋予胁迫耐受性以及探索构象动力学与酶功能之间的联系。然而，迄今为止，尚未对活性嗜热亲环蛋白进行充分的生物物理表征。在这里，我们确定了嗜碱芽孢杆菌的嗜热亲环蛋白（GeoCyp）的结构，使用一系列方法（包括基于化学位移的方法和在一定温度范围内的弛豫实验）表征了其在多个时间尺度上的动态运动，并测量催化活性在一定温度范围内进行比较，以比较其结构，动力学和功能与中温对应物人亲环蛋白A（CypA）的结构，动力学和功能。与大多数嗜热/嗜温菌对不同，与CypA相比，GeoCyp催化在低温下基本上没有受到损害，保持了其嗜温对应物活性的70％左右。底物结合体的检查揭示了一种机制，通过该机制，两个亲环蛋白可以通过改变动态环运动和关键残基来适应其环境，该关键残基起着钳子的作用，以差异性地调节CypA和GeoCyp中的底物结合。尽管高度依赖于结构，但根据高度结构相似性，两种蛋白之间的快速时间尺度（皮秒至纳秒级）动力学在很大程度上得以保留。较慢的（微秒）时标运动同样位于两种蛋白质中的相似区域，其大小存在一定差异，但在两种酶中均未表现出明显的温度依赖性。

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