Keywords

CDPK, TgCDPK1, TgCDPK3, CpCDPK, Calcium signaling, Activation mechanism, EF-hand, CAD, CH1, CH2, Structural biology

Reference

DOI: 10.1038/nsmb.1795

Abstract

Calcium-dependent protein kinases (CDPKs) are essential Ca2+ signaling effectors in plants and protists. They combine a CaMK-like kinase domain (KD) with a calcium-binding CDPK activation domain (CAD). By solving the crystal structures of autoinhibited (apo) and calcium-bound activated forms of TgCDPK1, TgCDPK3, and CpCDPK, this study uncovers the large-scale conformational rearrangements required for CDPK activation. Unlike typical CaMKs, CDPKs employ a calcium-triggered CAD rotation and remodeling mechanism, revealing an evolutionarily distinct but mechanistically elegant pathway for calcium signal transduction.

Notes

1. Structural Overview of CAD and Inhibitory Mechanism

  • CAD (~200 residues) acts as both autoinhibitor and Ca2+-sensor, wrapping around the kinase domain (KD) to prevent substrate access.
  • CH1 and CH2 helices form a dumbbell-shaped domain in the autoinhibited form, spanning the KD.
  • CH1 subdivided into functional segments:
    1. Autoinhibitory region (S/A322-Lys338): blocks substrate-binding site and engages KED triad (Lys-Glu135-Asp138), preventing ATP interaction.
    2. Hydrophobic residues Leu339, Met347, Leu351 mediate tight binding; conserved in CDPKs, reminiscent of CaMKII-CaM interaction motifs.
    3. EF-hand 1’s E-helix starts at Thr352–Asp368.

Key insight: CH1 secures the inactive conformation by interlocking with the KD, explaining why CDPKs are tightly controlled by CAD without requiring separate calmodulin.

2. Unique Autoinhibition Mechanisms Distinct from CaMK

  • CAD’s CH1 binds deeply into KD’s substrate site — unlike CaMKII’s flexible inhibition loop.
  • Conserved KED triad in KD, engaged by CH1 Lys338, stabilizes inactive conformation.
  • Insert in N-EF lobe of CAD (unique to some CDPKs, absent in CaM/TnC) helps lock KD in inactive form.
  • I212 in KD’s C-lobe interacts hydrophobically with CAD to lock activation segment in DFG-out (inactive).

3. Calcium-Triggered Activation: CAD Rearrangement

  • Upon Ca2+ binding, all four EF hands coordinate Ca2+, inducing dramatic refolding:

    • CH1 and CH2 unravel, intertwine, and relocate ~135° around KD.
    • EF-hands expose hydrophobic surfaces, aiding repositioning.
    • CH1 no longer blocks substrate site, thus relieving autoinhibition.

  • CAD in active state resembles a twisted, intertwined helical assembly—distinct from canonical CaM wrap-around mechanisms.

  • Twist-and-swing mechanism for CDPKs! Not simple loop displacement like CaMK, but entire domain rotation.

4. Implications for CDPK AutoPhosphorylation and Regulation

  • Autophosphorylation in CAD (seen in At and Pf CDPKs) might lock the kinase in active form, preventing return to autoinhibited state even if Ca2+ drops.
  • Possible S/T phosphorylation sites near CH1, between HXW motif and EF-hand, support this model.
  • Autophosphorylation potentially stabilizes the CAD post-activation, extending signal duration.

5. Functional Insights into Regulation

  • Full CAD required for activation—suggesting interplay of CH1, CH2, EF-hands critical.
  • No evidence that phosphorylation by other kinases is required—intrinsic Ca2+-binding and CAD rearrangement suffice.
  • CAD is a multifunctional domain: sensor, inhibitor, and allosteric activator—consolidating multiple roles into one elegant module.

6. Broader Implications

  • EF-hand based calcium regulation in CDPK is distinct from CaM-dependent pathways, showing evolutionary divergence.

  • Comparison to CaM and troponin C (TnC):

    • CAD is larger, with additional helical elements (CH1/CH2).
    • CH1-CH2 conformational plasticity allows unique activation mechanism.

  • CDPKs illustrate how domain fusion and novel structural arrangements evolve to suit specific signaling needs, particularly in parasitic organisms where rapid calcium responses are critical for lifecycle transitions (e.g., invasion, egress).

Figures

Inactive (apo) conformation of CDPKActivated (Ca2+-bound) conformation of CDPK
Figure 1. Inactive apo state of CDPK: CH1 blocks the KD substrate site.Figure 2. Activated CDPK: CAD rearranged, KD accessible for catalysis.

Take-home Messages

  • CDPK activation relies on a dramatic CAD rearrangement, not mere loop release.
  • CH1 and CH2 helices are central in mediating inhibition and activation through dynamic refolding and relocation.
  • Autophosphorylation may lock CDPK in the active conformation, modulating Ca2+ signaling duration.
  • The EF-hand system in CDPKs is tuned for integrated sensing and structural control, unlike modular CaM systems.

Final Thought

“In CDPKs, the calcium signal is read and executed through a masterful domain rearrangement, turning structural twists into cellular actions—showing how evolution shapes molecular machines for precision signaling."