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Elife. 2015 Sep 30;4. pii: e07574. doi: 10.7554/eLife.07574.

Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography.

Author information

1
Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, United States.
2
Department of Physics, Cornell University, Ithaca, United States.
3
Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, United States.
4
Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, United States.
5
Department of Molecular and Cellular Physiology, Stanford University, Stanford, United States.
6
Howard Hughes Medical Institute, Stanford University, Stanford, United States.
7
Linac Coherent Light Source, SLAC National Accelerator Laboratory, Menlo Park, United States.
8
Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States.
9
Department of Structural Biology, Stanford University, Stanford, United States.
10
Department of Photon Science, SLAC National Accelerator Laboratory, Menlo Park, United States.

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.

KEYWORDS:

biophysics; enzymology; human; protein dynamics; structural biology; x-ray free electron laser

PMID:
26422513
PMCID:
PMC4721965
DOI:
10.7554/eLife.07574
[Indexed for MEDLINE]
Free PMC Article

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