Keywords

EF-hand, Ca2+, Metal-ion Binding, Mg2+, Conformational Change, Cooperativity

Reference

DOI: 10.1042/BJ20070255

Abstract

EF-hand Ca²⁺-binding motifs play essential roles in eukaryotic signaling, found across a diverse protein superfamily.
Defined by a helix–loop–helix structure, they present semi-conserved Ca²⁺-binding ligands, though non-canonical variants exist.
EF-hands typically pair, facilitating positive cooperativity, enhancing Ca²⁺ sensitivity.
Ca²⁺ binding induces varied conformational responses, including target interaction site formation or structural stabilization.
The review addresses metal selectivity (Ca²⁺ vs. Mg²⁺), binding cooperativity, and conformational diversity, providing a structural and biophysical perspective on EF-hand proteins.

Notes

1. EF-hand Overview

  • Helix-loop-helix motif identified in parvalbumin, crucial for Ca²⁺ sensing and buffering.
  • Two classes:
    • Ca²⁺ sensors (e.g., calmodulin) with conformational change for signaling.
    • Ca²⁺ buffers (e.g., calbindin) modulating intracellular Ca²⁺ levels without major structural shifts.
  • Binding specificity for Ca²⁺ over Mg²⁺, despite high cellular Mg²⁺ concentration, achieved via geometry and dehydration energy differences.

2. Structural Principles of EF-hand

  • Canonical EF-loops:
    • 12 residues, coordinating Ca²⁺ in pentagonal bipyramidal geometry (7 oxygen ligands).
    • Five ligands from loop, two from adjacent residues.
    • Aspartic acid often involved due to smaller side chain, favoring tight coordination.
  • Non-canonical EF-loops:
    • Modified ligand set or loop length (e.g., Asp replacing Glu at position 12, use of water molecules).
    • Alternative coordination geometries (e.g., octahedral in ALG-2).

3. EF-hand Pairing and Cooperativity

  • EF-hands usually paired, forming four-helix bundles with binding sites ~11 Å apart.
  • Pairs can exhibit positive cooperativity, enhancing Ca²⁺ sensitivity—especially in sensors like CaM, TnC.
  • Antiparallel β-sheets and interhelical contacts stabilize pairs, mediating binding communication.
  • Positive cooperativity mechanisms:
    • First Ca²⁺ binding stabilizes the β-sheet, creating a favorable environment for second binding.
    • Structural reorganization of helices enhances second site affinity.

4. Conformational Dynamics upon Ca²⁺ Binding

  • Conformational changes vary by protein function:
    • Sensors: Transition from closed to open, exposing hydrophobic sites for target binding (e.g., CaM).
    • Buffers: Minimal structural change; maintain closed conformation (e.g., calbindin).
    • Others like S100 adjust interhelical angles; recoverin undergoes domain rotation.
  • Dynamic plasticity allows flexibility for target interaction, stabilized by Ca²⁺.

5. Ca²⁺/Mg²⁺ Selectivity and Dual Metal Binding

  • EF-hands distinguish Ca²⁺ vs. Mg²⁺ based on:
    • Coordination geometry: Ca²⁺ accommodates flexible arrangements; Mg²⁺ requires rigid octahedral coordination.
    • Dehydration cost: Mg²⁺ harder to desolvate, less favorable.
    • Ligand flexibility: Twelfth residue (often Glu) adapts to bind Ca²⁺ (bidentate) or Mg²⁺ (monodentate).
  • Mg²⁺ roles:
    • Maintain structure in absence of Ca²⁺ (e.g., parvalbumin, calmodulin).
    • Modulate Ca²⁺ affinity and signaling responsiveness.
    • Functional roles: In DREAM (DNA binding), CaV1.2 channel (stress response inhibition).

6. Ca²⁺ Binding Mechanisms and Energetics

  • Entropic contribution: Water release upon Ca²⁺ binding increases system entropy.
  • Enthalpic contribution:
    • Electrostatic interactions between Ca²⁺ and ligands.
    • Hydrogen bonds stabilizing the bound state.
    • Helical packing enhancing the binding site’s rigidity.
  • Stability differences: Unstable apo states drive high Ca²⁺ affinity via larger ΔG.
  • Example: Calmodulin N-/C-terminal domains show differing Ca²⁺ affinities due to intrinsic stability.

7. Functional Implications of Cooperativity and Target Interactions

  • Positive cooperativity enhances sensitivity to Ca²⁺ concentration changes—crucial for rapid signaling.
  • Cooperativity mechanisms:
    • β-sheet formation stabilizes first binding.
    • Interhelical interactions transmit binding state information.
  • Independent vs. cooperative binding:
    • Independent: CaVP, CIB—each EF-hand binds Ca²⁺ independently.
    • Sequential cooperative: sTnC, recoverin—first site binding enhances second site affinity.
  • Target interactions modulate affinity:
    • Target binding stabilizes Ca²⁺-bound state, reduces dissociation (e.g., CaM).
    • Can either enhance or reduce affinity depending on regulatory needs.

8. RD’s Thoughts and Takeaways

  • RD appreciates the mechanistic depth in explaining EF-hand diversity and adaptability.
  • The balance of entropic/enthalpic contributions makes EF-hands finely tuned Ca²⁺ sensors.
  • The discussion on Mg²⁺ competition and dual-binding sites is particularly insightful for interpreting EF-hand function in different environments.
  • Structural flexibility and conformational plasticity are crucial for EF-hands acting as multi-functional signaling hubs.
  • RD finds the exploration of positive cooperativity and its structural basis useful for understanding Ca²⁺ signal transduction.

Take-home Messages

  • EF-hands are versatile, dynamic Ca²⁺-binding motifs, integral to cellular signaling.
  • Structural design (canonical/non-canonical loops, helix packing) defines Ca²⁺ affinity and response.
  • Metal-ion selectivity (Ca²⁺ vs. Mg²⁺) and positive cooperativity enable precise control over signaling pathways.
  • Conformational changes upon Ca²⁺ binding drive target recognition and signal relay.
  • Interplay of structural, energetic, and dynamic factors underlies EF-hand functional diversity.