Holliday Michael J, Camilloni Carlo, Armstrong Geoffrey S, Isern Nancy G, Zhang Fengli, Vendruscolo Michele, Eisenmesser Elan Z
†Department of Biochemistry and Molecular Genetics, University of Colorado Denver, 12801 East 17th Avenue, Aurora, Colorado 80045, United States.
‡Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, U.K.
Biochemistry. 2015 May 26;54(20):3207-17. doi: 10.1021/acs.biochem.5b00263. Epub 2015 May 14.
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 ~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.
嗜热蛋白因其在高温下具有高稳定性和功能性，在研究和工业应用中得到了广泛应用，同时也为我们理解蛋白质折叠、稳定性、动力学和功能提供了有价值的见解。亲环蛋白是一类普遍表达的肽基脯氨酰异构酶家族，具有一系列生物学功能和疾病关联，已被用于赋予应激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，尚未对任何活性嗜热亲环蛋白进行全面的生物物理表征。在此，我们确定了嗜碱芽孢杆菌嗜热亲环蛋白（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系列温度下的弛豫实验）表征了其在多个时间尺度上的动态运动，并测量了一系列温度下的催化活性，以将其结构、动力学和功能与嗜温对应物人亲环蛋白A（CypA）进行比较。与大多数嗜热菌/嗜温菌对不同，与CypA相比，GeoCyp在低温下的催化作用并未受到实质性损害，保留了其嗜温对应物约70%的活性。对底物结合整体的研究揭示了一种机制，通过该机制，两种亲环蛋白可能通过改变动态环运动以及一个关键残基来适应其环境，该关键残基在CypA和GeoCyp中作为一个夹子来差异调节底物结合。尽管两种蛋白质在温度依赖性方面存在差异，但根据高度的结构相似性，它们在快速时间尺度（皮秒到纳秒）的动力学在很大程度上是保守的。较慢（微秒）时间尺度的运动同样定位于两种蛋白质中的相似区域，其幅度存在一些变化，但在两种酶中均未表现出明显的温度依赖性。
