Keywords

CDPK, CAD, CpCDPK3, PfCDPK, Apicomplexa, Calcium activation, EF-hand, Structural mechanism, Conformational change

Reference

DOI: 10.1002/prot.22919

Abstract

Building on earlier work (NSMB, 2010), this study extends structural insights into apicomplexan CDPK activation, focusing on isolated CADs of Cryptosporidium parvum (CpCDPK3) and Plasmodium falciparum (PfCDPK3). Here, the authors present intermediate CAD conformations, identify conserved structural motifs, and analyze mechanistic elements governing Ca2+-triggered activation. The work proposes a conserved mechanism across CDPK family members, potentially extending to plant CDPKs, involving a collapse of CAD helices (CH1/CH2) and EF-hand refolding upon calcium binding.

Notes

1. CAD Conformational Dynamics and Activation

  • In inactive form (apo): CAD is extended; CH1 wraps behind KD as CH1-1 (autoinhibitory helix).
  • Upon Ca2+ binding: CAD collapses into a compact helical bundle, shifting to the distal side of KD, relieving inhibition.
  • 🌸 Key insight: Calcium triggers a unique “collapse-and-lock” mechanism, distinct from CaM “wrap-around” mode.

2. CH1/CH2 Roles and Intrinsic Regulation

  • CH1 is subdivided:

    • CH1-1: Autoinhibitory sequence, crucial for blocking KD.
    • CH1-2: Contains pseudosubstrate motif, with conserved KL motif; binds KED triad in KD. Mutation in AtCDPK1 (L→A) reduces activation.
    • CH1-3: Interface for CH1-1/CH2 interaction, completing a circular clasp around KD.

  • 🌸 Conserved interactions (GKH(AL)TGALGNMKKF motif) stabilize the inactive state, and unlock upon activation.

3. EF-Hand Specificity and Divergence

  • EF1-EF4: Canonical calcium-binding sites, but only 3 Ca2+ bound in PfCDPK3 due to Q replacing E in EF1 — partial loop destabilization, hydrogen bonds compensate.
  • Ca2+ occupancy sequence: Likely C-lobe first (high affinity) → N-lobe later, consistent with graded activation threshold.

🌸 Suggests PfCDPK3 may respond to lower Ca2+ signals, fine-tuning activation threshold.

4. N-terminal Latch: A Regulatory Element?

  • An N-terminal latch in CAD engages KD in inactive state, releasing upon activation.
  • Contains potential myristoylation motifs in some CDPKs — membrane targeting or localization?

🌸 Interesting layer of regulation yet to be fully understood.

5. Sequence Conservation and Functional Insights

  • EF-hand core residues highly conserved across Apicomplexa and plants.
  • Surface residues diverge, likely for organism-specific tuning.
  • Phe333 (interface) and Tyr424 (absolutely conserved) are key allosteric residues.
  • CH2 helix aligns well with CaM central linker, indicating possible functional analog.

6. Intermediates and Refolding Mechanism

  • CpCDPK3_CAD and PfCDPK3_CAD captured intermediate conformations—support stepwise refolding:
    1. EF-hands bind Ca2+, initiating hydrophobic residue exposure.
    2. CH2 unwinds, engaging CH1 and EF lobes.
    3. Autoinhibitory CH1-1 released from KD.
    4. Final collapse into activated CAD.

🌸 CAD refolding is a “domino-like” cascade, stabilized step-by-step by Ca2+ and intramolecular interactions.

7. Comparisons to Plant CDPK and Functional Predictions

  • Plant CDPKs (e.g., AtCDPK1) share similar motifs (e.g., KL, CH1-2 pseudomotif, core EF residues).
  • Differences: No CH1-1 in AtCDPK, suggesting distinct regulatory nuances.
  • AutoP sites identified in CH1-2 and CAD, potentially locking active state or modulating return to inactive.
  • 🌸 Possible common refolding path but distinct thresholds and kinetics depending on species.

8. Cool Experimental Insights

  • Calcium binding to EF2 may “prime” EF1 via H-bonds—cooperativity in Ca2+ binding.
  • N-lobe twist and CH1 disengagement as key structural events leading to activation.
  • Possible fine-tuning of activation via CH1/CH2 phosphorylation.

Figures


Figure: A pic from the paper (Click to enlarge)

Take-home Messages

  • CAD-mediated activation of CDPKs is a universal mechanism in Apicomplexa and possibly conserved in plants.
  • Activation proceeds via coordinated EF-hand Ca2+ binding and sequential helical rearrangement.
  • CH1/CH2 act as internal target-like modules, regulating KD access.
  • Variations in EF-hand and loop composition fine-tune activation thresholds across species.
  • 🌸 CDPKs are elegant allosteric switches, integrating calcium sensing and auto-regulation through a tightly coupled structural relay.

🌿 Final Thought

🌸 “From binding calcium to flipping activation switches, CDPKs show how nature builds precise regulatory machines, evolving distinct paths to interpret universal signals like calcium."