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

Journal name:
Nature Structural & Molecular Biology
Volume:
17,
Pages:
596–601
Year published:
DOI:
doi:10.1038/nsmb.1795
Received
Accepted
Published online

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.

At a glance

Figures

  1. Effect of Ca2+ on CDPKs.
    Figure 1: Effect of Ca2+ on CDPKs.

    (a) All three enzymes were tested for activity using Syntide-2 as a substrate, and their activities were found to increase with increasing Ca2+ concentrations (see Online Methods). (b) Calcium in-out-in assay. TgCDPK1 and TgCDPK3 partially lost activity after EDTA treatment (relative to activity level before treatment), whereas CpCDPK1 became virtually inactive (samples labeled E). All three regained activity when calcium was added back (labeled E-Ca). Error bars represent s.d. (n = 3).

  2. Crystal structures of apicomplexan CDPKs.
    Figure 2: Crystal structures of apicomplexan CDPKs.

    (ad) The first four structures (ad) are oriented by aligning their kinase domains. The kinase domain is shown in gray in all cases, with the CAD shown in color. The CH1 and CH2 helices are highlighted in cyan and pale magenta, respectively, with these helices broken into three segments in the activated conformation (a-CpCDPK1 and a-TgCDPK1). The autoinhibitory segment is highlighted in a red box. The N-terminal latch in TgCDPK1 is shown in green. Calcium ions are shown as green spheres, with those hidden in the protein structures indicated by green arrows. (a) Structure of i-TgCDPK1. A metal ion was observed in three of the four EF hands. The coordination of each did not match that of Ca2+ or Mg2+. The pseudosubstrate autoinhibitory triad is highlighted. (b) Structure of i-TgCDPK3. A Mg2+ ion was found in one of the EF hands in the C terminus. (c) Structure of a-TgCDPK1 with four Ca2+ ions bound (green spheres). (d) Structure of a-CpCDPK1 with four Ca2+ ions bound. (e) Cartoon schematic of i-TgCDPK1 and a-TgCDPK1 superimposed with the kinase domain aligned, showing that the CAD has translocated from one side to the other. (f) Cartoon schematic of i-TgCDPK1 and a-TgCDPK1 superimposed with the kinase domain aligned, as seen looking 'down' at the β lobe and showing the extent the translocation of the CAD around the kinase domain.

  3. Kinase domain-CAD interfaces.
    Figure 3: Kinase domain–CAD interfaces.

    (a) The insert in the N-EF lobe is highlighted by a black circle, showing that it is in close proximity to the kinase domain in the i-TgCDPK1 structure. (b) The interface between the insert in the N-EF lobe and the kinase domain (again highlighted in black circle) is seen from 'the other side' of the i-TgCDPK1 structure. Also shown is the brown activation segment locked in its inactive conformation by the engagement of Ile212 (highlighted in green circle) in a hydrophobic pocket in the surface of the CAD. (c) In the inactivated conformation, the CH1 helix (cyan) in the CAD has a number of hydrophobic residues buried in the amphipathic cleft at the base of the C lobe of the kinase domain. (d) The helix αD undergoes a noticeable shift from its position in i-TgCDPK1 (blue) to a new position in a-TgCDPK1 (green), where it closes the cleft occupied by CH1 in the inactive conformation. This shift is made possible by the translocation of the CAD. (e) The key interfaces between the kinase domain and the CAD in the activated conformation of TgCDPK1 are shown. The green helix in the N terminus is latched into the CAD. The activation segment (brown) is free to move into its active conformation.

  4. Schematic representing the activation of a canonical CDPK.
    Figure 4: Schematic representing the activation of a canonical CDPK.

    (a) Cartoon schematic showing the inactivated (left) and activated (right) structures of TgCDPK1, with helices rendered as cylinders. The color scheme follows that used in Figures 2 and 3. (b) Cartoon schematic showing only the CH1 and CH2 helices in the calcium-absent (left) and calcium-loaded forms of the CAD (right). A new color scheme is used here to facilitate matching of the corresponding segments in the two states. The gray segments are those that unwind and bend in the transition from the inhibited state to the activated state. The residue sequences of the two helices are also shown in the same color scheme. (c) Simplified schematic of the CAD in the inactivated (left) and activated (right) states. The color scheme follows that used in b. The green circles represent the calcium ions. The large circles represent the EF lobes. The autoinhibitory segments are indicated as AS.

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References

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Author information

Affiliations

  1. Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.

    • Amy K Wernimont,
    • Jennifer D Artz,
    • Patrick Finerty Jr,
    • Yu-Hui Lin,
    • Mehrnaz Amani,
    • Abdellah Allali-Hassani,
    • Guillermo Senisterra,
    • Masoud Vedadi,
    • Wolfram Tempel,
    • Farrell Mackenzie,
    • Irene Chau &
    • Raymond Hui
  2. Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA.

    • Sebastian Lourido &
    • L David Sibley

Contributions

Y.L. and M.A. purified and crystallized the proteins and performed MS analyses; A.K.W. and W.T. collected and processed X-ray data; A.K.W., J.D.A. and R.H. designed the experiments and analyzed the results; A.K.W. refined and analyzed the structural models; F.M. cloned the constructs; S.L. and L.D.S. identified the entry clone for TgCDPK1; L.D.S. provided functional analysis; P.F. Jr., A.A.-H., G.S. and I.C. conducted various assays on the proteins and analyzed the results; M.V. was involved in analysis of assay results.

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The authors declare no competing financial interests.

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