Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography
Category
Published on
Type
journal-article
Author
Daniel A Keedy and Lillian R Kenner and Matthew Warkentin and Rahel A Woldeyes and Jesse B Hopkins and Michael C Thompson and Aaron S Brewster and Andrew H Van Benschoten and Elizabeth L Baxter and Monarin Uervirojnangkoorn and Scott E McPhillips and Jinhu Song and Roberto Alonso-Mori and James M Holton and William I Weis and Axel T Brunger and S Michael Soltis and Henrik Lemke and Ana Gonzalez and Nicholas K Sauter and Aina E Cohen and Henry van den Bedem and Robert E Thorne and James S Fraser
Citation
Keedy, D.A. et al., 2015. Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography. eLife, 4. Available at: http://dx.doi.org/10.7554/elife.07574.
Abstract
Determining the interconverting conformations of dynamic proteins in atomic detail is a major challenge for structural biology. Conformational heterogeneity in the active site of the dynamic enzyme cyclophilin A (CypA) has been previously linked to its catalytic function, but the extent to which the different conformations of these residues are correlated is unclear. Here we compare the conformational ensembles of CypA by multitemperature synchrotron crystallography and fixed-target X-ray free-electron laser (XFEL) crystallography. The diffraction-before-destruction nature of XFEL experiments provides a radiation-damage-free view of the functionally important alternative conformations of CypA, confirming earlier synchrotron-based results. We monitored the temperature dependences of these alternative conformations with eight synchrotron datasets spanning 100-310 K. Multiconformer models show that many alternative conformations in CypA are populated only at 240 K and above, yet others remain populated or become populated at 180 K and below. These results point to a complex evolution of conformational heterogeneity between 180-–240 K that involves both thermal deactivation and solvent-driven arrest of protein motions in the crystal. The lack of a single shared conformational response to temperature within the dynamic active-site network provides evidence for a conformation shuffling model, in which exchange between rotamer states of a large aromatic ring in the middle of the network shifts the conformational ensemble for the other residues in the network. Together, our multitemperature analyses and XFEL data motivate a new generation of temperature- and time-resolved experiments to structurally characterize the dynamic underpinnings of protein function.
DOI
Funding
NSF-STC Biology with X-ray Lasers (NSF-1231306)
