Abstract
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.