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

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 inmore » the middle of the network shifts the conformational ensemble for the other residues in the network. Altogether, 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.« less

Authors:
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Publication Date:
Research Org.:
SLAC National Accelerator Lab., Menlo Park, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1233981
Alternate Identifier(s):
OSTI ID: 1233982; OSTI ID: 1240121
Grant/Contract Number:  
AC02-76SF00515; GM103485; DMR1332208; STC1231306; GM110580; OD009180; SLAC-LDRD-0014-13-2; MCB1330685; GM094586; GM103393; GM102520; GM095887
Resource Type:
Published Article
Journal Name:
eLife
Additional Journal Information:
Journal Name: eLife Journal Volume: 4; Journal ID: ISSN 2050-084X
Publisher:
eLife Sciences Publications, Ltd.
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Keedy, Daniel A., Kenner, Lillian R., Warkentin, Matthew, Woldeyes, Rahel A., Hopkins, Jesse B., Thompson, Michael C., Brewster, Aaron S., Van Benschoten, Andrew H., Baxter, Elizabeth L., Uervirojnangkoorn, Monarin, McPhillips, Scott E., Song, Jinhu, Alonso-Mori, Roberto, Holton, James M., Weis, William I., Brunger, Axel T., Soltis, S. Michael, Lemke, Henrik, Gonzalez, Ana, Sauter, Nicholas K., Cohen, Aina E., van den Bedem, Henry, Thorne, Robert E., and Fraser, James S.. Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography. United States: N. p., 2015. Web. https://doi.org/10.7554/eLife.07574.
Keedy, Daniel A., Kenner, Lillian R., Warkentin, Matthew, Woldeyes, Rahel A., Hopkins, Jesse B., Thompson, Michael C., Brewster, Aaron S., Van Benschoten, Andrew H., Baxter, Elizabeth L., Uervirojnangkoorn, Monarin, McPhillips, Scott E., Song, Jinhu, Alonso-Mori, Roberto, Holton, James M., Weis, William I., Brunger, Axel T., Soltis, S. Michael, Lemke, Henrik, Gonzalez, Ana, Sauter, Nicholas K., Cohen, Aina E., van den Bedem, Henry, Thorne, Robert E., & Fraser, James S.. Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography. United States. https://doi.org/10.7554/eLife.07574
Keedy, Daniel A., Kenner, Lillian R., Warkentin, Matthew, Woldeyes, Rahel A., Hopkins, Jesse B., Thompson, Michael C., Brewster, Aaron S., Van Benschoten, Andrew H., Baxter, Elizabeth L., Uervirojnangkoorn, Monarin, McPhillips, Scott E., Song, Jinhu, Alonso-Mori, Roberto, Holton, James M., Weis, William I., Brunger, Axel T., Soltis, S. Michael, Lemke, Henrik, Gonzalez, Ana, Sauter, Nicholas K., Cohen, Aina E., van den Bedem, Henry, Thorne, Robert E., and Fraser, James S.. Wed . "Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography". United States. https://doi.org/10.7554/eLife.07574.
@article{osti_1233981,
title = {Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography},
author = {Keedy, Daniel A. and Kenner, Lillian R. and Warkentin, Matthew and Woldeyes, Rahel A. and Hopkins, Jesse B. and Thompson, Michael C. and Brewster, Aaron S. and Van Benschoten, Andrew H. and Baxter, Elizabeth L. and Uervirojnangkoorn, Monarin and McPhillips, Scott E. and Song, Jinhu and Alonso-Mori, Roberto and Holton, James M. and Weis, William I. and Brunger, Axel T. and Soltis, S. Michael and Lemke, Henrik and Gonzalez, Ana and Sauter, Nicholas K. and Cohen, Aina E. and van den Bedem, Henry and Thorne, Robert E. and Fraser, James S.},
abstractNote = {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. Altogether, 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 = {10.7554/eLife.07574},
journal = {eLife},
number = ,
volume = 4,
place = {United States},
year = {2015},
month = {9}
}

Journal Article:
Free Publicly Available Full Text
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https://doi.org/10.7554/eLife.07574

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