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Title: Structures of apicomplexan calcium-dependent protein kinases reveal mechanism of activation by calcium

Abstract

Calcium-dependent protein kinases (CDPKs) have pivotal roles in the calcium-signaling pathway in plants, ciliates and apicomplexan parasites and comprise a calmodulin-dependent kinase (CaMK)-like kinase domain regulated by a calcium-binding domain in the C terminus. To understand this intramolecular mechanism of activation, we solved the structures of the autoinhibited (apo) and activated (calcium-bound) conformations of CDPKs from the apicomplexan parasites Toxoplasma gondii and Cryptosporidium parvum. In the apo form, the C-terminal CDPK activation domain (CAD) resembles a calmodulin protein with an unexpected long helix in the N terminus that inhibits the kinase domain in the same manner as CaMKII. Calcium binding triggers the reorganization of the CAD into a highly intricate fold, leading to its relocation around the base of the kinase domain to a site remote from the substrate binding site. This large conformational change constitutes a distinct mechanism in calcium signal-transduction pathways.

Authors:
; ; ; ; ; ; ; ; ; ; ; ; ;  [1];  [2]
  1. (Toronto)
  2. (
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
USDOE
OSTI Identifier:
1002708
Resource Type:
Journal Article
Resource Relation:
Journal Name: Nat. Struct. Mol. Biol.; Journal Volume: 17; Journal Issue: 2010
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; 99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE; CALCIUM; CALMODULIN; CONFORMATIONAL CHANGES; PARASITES; PHOSPHOTRANSFERASES; PROTEINS; SUBSTRATES

Citation Formats

Wernimont, Amy K, Artz, Jennifer D., Jr, Patrick Finerty, Lin, Yu-Hui, Amani, Mehrnaz, Allali-Hassani, Abdellah, Senisterra, Guillermo, Vedadi, Masoud, Tempel, Wolfram, Mackenzie, Farrell, Chau, Irene, Lourido, Sebastian, Sibley, L. David, Hui, Raymond, and WU-MED). Structures of apicomplexan calcium-dependent protein kinases reveal mechanism of activation by calcium. United States: N. p., 2010. Web. doi:10.1038/nsmb.1795.
Wernimont, Amy K, Artz, Jennifer D., Jr, Patrick Finerty, Lin, Yu-Hui, Amani, Mehrnaz, Allali-Hassani, Abdellah, Senisterra, Guillermo, Vedadi, Masoud, Tempel, Wolfram, Mackenzie, Farrell, Chau, Irene, Lourido, Sebastian, Sibley, L. David, Hui, Raymond, & WU-MED). Structures of apicomplexan calcium-dependent protein kinases reveal mechanism of activation by calcium. United States. doi:10.1038/nsmb.1795.
Wernimont, Amy K, Artz, Jennifer D., Jr, Patrick Finerty, Lin, Yu-Hui, Amani, Mehrnaz, Allali-Hassani, Abdellah, Senisterra, Guillermo, Vedadi, Masoud, Tempel, Wolfram, Mackenzie, Farrell, Chau, Irene, Lourido, Sebastian, Sibley, L. David, Hui, Raymond, and WU-MED). 2010. "Structures of apicomplexan calcium-dependent protein kinases reveal mechanism of activation by calcium". United States. doi:10.1038/nsmb.1795.
@article{osti_1002708,
title = {Structures of apicomplexan calcium-dependent protein kinases reveal mechanism of activation by calcium},
author = {Wernimont, Amy K and Artz, Jennifer D. and Jr, Patrick Finerty and Lin, Yu-Hui and Amani, Mehrnaz and Allali-Hassani, Abdellah and Senisterra, Guillermo and Vedadi, Masoud and Tempel, Wolfram and Mackenzie, Farrell and Chau, Irene and Lourido, Sebastian and Sibley, L. David and Hui, Raymond and WU-MED)},
abstractNote = {Calcium-dependent protein kinases (CDPKs) have pivotal roles in the calcium-signaling pathway in plants, ciliates and apicomplexan parasites and comprise a calmodulin-dependent kinase (CaMK)-like kinase domain regulated by a calcium-binding domain in the C terminus. To understand this intramolecular mechanism of activation, we solved the structures of the autoinhibited (apo) and activated (calcium-bound) conformations of CDPKs from the apicomplexan parasites Toxoplasma gondii and Cryptosporidium parvum. In the apo form, the C-terminal CDPK activation domain (CAD) resembles a calmodulin protein with an unexpected long helix in the N terminus that inhibits the kinase domain in the same manner as CaMKII. Calcium binding triggers the reorganization of the CAD into a highly intricate fold, leading to its relocation around the base of the kinase domain to a site remote from the substrate binding site. This large conformational change constitutes a distinct mechanism in calcium signal-transduction pathways.},
doi = {10.1038/nsmb.1795},
journal = {Nat. Struct. Mol. Biol.},
number = 2010,
volume = 17,
place = {United States},
year = 2010,
month = 9
}
  • Calcium-dependent protein kinases (CDPKs) comprise the major group of Ca 2+-regulated kinases in plants and protists. It has long been assumed that CDPKs are activated, like other Ca 2+-regulated kinases, by derepression of the kinase domain (KD). However, we found that removal of the autoinhibitory domain from Toxoplasma gondii CDPK1 is not sufficient for kinase activation. From a library of heavy chain-only antibody fragments (VHHs), we isolated an antibody (1B7) that binds TgCDPK1 in a conformation-dependent manner and potently inhibits it. We uncovered the molecular basis for this inhibition by solving the crystal structure of the complex and simulating, throughmore » molecular dynamics, the effects of 1B7–kinase interactions. In contrast to other Ca 2+-regulated kinases, the regulatory domain of TgCDPK1 plays a dual role, inhibiting or activating the kinase in response to changes in Ca 2+ concentrations. We propose that the regulatory domain of TgCDPK1 acts as a molecular splint to stabilize the otherwise inactive KD. This dependence on allosteric stabilization reveals a novel susceptibility in this important class of parasite enzymes.« less
  • The soluble and membrane-bound forms of the calcium-dependent protein kinase from barley leaves (Hordeum vulgare L. cv. Borsoy) have been partially purified and compared. Both forms showed an active polypeptide of 37 kilodaltons on activity gels with incorporated histone as substrate. They eluted from chromatofocusing columns at an identical isoelectric point of pH 4.25 {plus minus} 0.2, and also comigrated on several other chromatographic affinity media including Matrex Gel Blue A, histone-agarose, phenyl-Sepharose, and heparin-agarose. Both activities comigrated with chicken ovalbumin during gel filtration through Sephacryl S-200, indicating a native molecular mass of 45 kilodaltons. The activities share a numbermore » of enzymatic properties including salt and pH dependence, free calcium stimulation profile, substrate specificity, and Km values. The soluble activity was shown to bind to artificial lipid vesicles. These data suggest strongly that the soluble and membrane-bound calcium-dependent protein kinases from barley are very closely related or even identical.« less