Keywords

CDPK, TgCDPK1, Calcium signaling, Allosteric activation, Calmodulin-like domain, CAD, EF-hand, VHH, Splint model, Protein kinase stability

Reference

DOI: 10.1073/pnas.1505914112

Abstract

CDPKs (Calcium-dependent protein kinases) are the main calcium-regulated kinases in plants and protists. Traditionally, CDPK activation was thought to occur through removal of autoinhibition, similar to other Ca2+-dependent kinases (e.g., CaMKs). However, in Toxoplasma gondii CDPK1 (TgCDPK1), removing the regulatory domain does not activate the kinase. Instead, TgCDPK1 requires allosteric stabilization by its regulatory domain (CAD). A heavy chain-only antibody fragment (VHH 1B7) identified as a conformation-specific inhibitor further reveals that the regulatory domain functions as a “molecular splint” to stabilize TgCDPK1’s active state. This dual role of CAD (both inhibition and activation) reflects a novel regulatory mechanism unique to apicomplexan CDPKs.

Notes

1. Challenge to Classical Activation Model: CAD is Necessary for Activation

  • Deletion of CAD (residues 1–314 or 1–336) does NOT activate TgCDPK1—contrary to expectations based on CaMKs.
  • 3C-protease cleavage that physically separates KD and CAD also abolishes activity, emphasizing that tethering of CAD to KD is crucial for function.
  • CAD acts as an allosteric “splint” to stabilize and correctly align the KD for catalysis.
  • Key insight: CDPKs like TgCDPK1 are NOT simply inhibited kinases released by Ca2+, but require positive stabilization through domain interactions.

2. VHH 1B7 as a Conformation-dependent Inhibitor and Reporter

  • 1B7 VHH antibody selectively binds the Ca2+-bound active conformation of TgCDPK1, inhibiting its kinase activity.
  • 1B7 binding is Ca2+-dependent, shown by SEC and IP assays—released upon Ca2+ chelation (EGTA).
  • 1B7 inhibits TgCDPK1 and related TgCDPK3 with IC50 ~40 nM (complete inhibition at 1:1 ratio).
  • Recognizes EF-hand 1 and 2 of CAD and interacts with Ca2+ coordination residues (e.g., Lys350, Thr361).
  • VHH as a tool for probing active conformations! Potential use for capturing transient active states of kinases?

3. Dual Role of CAD: Allosteric Activation + Autoinhibition

  • CAD functions both as an inhibitor and activator:
    • Blocks active site in absence of Ca2+ (autoinhibition).
    • Stabilizes active KD conformation in presence of Ca2+ (allosteric activation).
  • N-terminal α-helix (J domain + EF1 helix) forms a single elongated helix occluding the active site (autoinhibited state, 3KU2).
  • In Ca2+-bound state (3HX4), CAD undergoes structural rearrangement, enabling KD activation.
  • 1B7-bound structure (4YGA) reveals key interactions that “freeze” CAD in the Ca2+-bound conformation—preventing catalysis.

4. Molecular Dynamics: CAD Stabilizes Active Kinase Domain

  • MD simulations show that KD alone is intrinsically unstable:
    • Loses active conformation (αC helix shifts out; R-spine misaligned).
  • CAD is necessary to stabilize KD:
    • Acts as a mechanical splint, supporting αC helix and R-spine (hydrophobic residues: Leu103, Leu114, His172, Phe196).
    • Without CAD: KD collapses into inactive “αC-out” conformation.

First-time evidence that CDPK KD is inherently inactive, needing a regulatory domain for functional alignment—opposite to CaMKs where KD is active when free.

5. Structural and Functional Specificity of the CAD

  • CAD interacts extensively with KD, tethering to both lobes.
  • Torque generated by CAD rearrangement (upon Ca2+ binding) may drive activation.
  • Increasing the flexibility of the CAD-KD tether (longer linker) reduces kinase activity—suggesting that precise geometry is essential.

6. Role of N-terminal Extension in Activation

  • N-terminal residues (F39, V40) critical for TgCDPK1 activity:
    • F39A completely abolishes activity.
    • V40A shifts EC50 for Ca2+ activation.
  • N-terminal deletion mutants lack kinase activity in vitro and fail to complement CDPK1 function in vivo (parasite invasion).

N-terminal extensions (in addition to CAD) contribute to fine-tuning allosteric control in CDPKs—possible tethering/anchoring roles?

Take-home Messages

  • TgCDPK1 is activated allosterically, NOT merely by release of an autoinhibitory domain.
  • The CAD (regulatory domain) stabilizes KD active conformation, acting like a splint.
  • VHH 1B7 serves as a conformation-dependent probe and inhibitor, valuable for studying kinase dynamics.
  • First direct evidence that apicomplexan CDPKs (unlike animal CaMKs) require regulatory domain for activation, challenging existing models.
  • Important implications for targeting CDPKs in parasites (e.g., Toxoplasma, Plasmodium)—disrupting CAD-KD interface may block kinase activity.

Final Thought

“Stabilization, not liberation”—the CAD in TgCDPK1 shows that some kinases need support, not just release, to function. A paradigm shift in understanding calcium-regulated kinases in protists, and a fascinating case of allosteric precision engineering by nature."