Keywords

EF-hand, Ca2+, Calcium-binding, Conformational Change, EFbeta-scaffold

Reference

DOI: 10.1016/j.jmb.2006.03.066

Abstract

EF-hand calcium-binding proteins are essential for regulating various cellular functions.
They display diverse structures, Ca²⁺-binding modes, and target interactions.
The two-step Ca²⁺-binding mechanism governed by the EFbeta-scaffold underlies conformational responses.
Key to this mechanism is the central β-scaffold connecting the Ca²⁺-binding loops, coordinating structural dynamics between variable N-terminal and conserved C-terminal parts of EF-hand motifs.
Other factors like interhelical contacts, loop length, and energy balance fine-tune the Ca²⁺-induced conformational changes.

Notes

1. General EF-hand Structural Principles

  • EF-hand motif: Conserved helix-loop-helix unit, initially identified in parvalbumin.
  • Canonical EF-hand loops:
    • 12-residue loops coordinating Ca²⁺ via pentagonal bipyramid geometry (7 oxygen atoms).
    • N-terminal loop: Variable, flexible.
    • C-terminal loop: Conserved, stabilizes Ca²⁺ coordination and helix formation.
  • Glutamate at -Z position: Crucial for Ca²⁺ coordination; occasionally replaced in noncanonical motifs (e.g., by Asp).

2. Pairing and Dimerization

  • EF-hands often occur in pairs, stabilized via β-sheet hydrogen bonds and hydrophobic interactions.
  • “Odd” and “Even” EF-hands: Non-identical but complementary within pairs.
  • Homodimers (e.g., S100) and heterodimers (e.g., Calmodulin, Troponin C) enable functional diversity.
  • Pairing allows fine-tuning of Ca²⁺-affinity and conformational response.

3. Conformational Changes Upon Ca²⁺ Binding

  • Regulatory EF-hand proteins:
    • Undergo domain opening, exposing hydrophobic surfaces for target binding.
    • Calmodulin (CaM), Troponin C (TnC): Classic examples.
  • Non-sensor EF-hand proteins (e.g., Calbindin D9k): Act as buffers, remain closed upon Ca²⁺ binding.
  • Mechanism:
    • Ca²⁺ binding relieves strain, driving domain opening in sensor proteins.
    • Non-sensor proteins are energetically stable in closed form.

4. EFbeta-scaffold and Two-step Ca²⁺-Binding Mechanism

  • Step 1: Ca²⁺ binds N-terminal loop, stabilizing the “incoming” helix.
  • Step 2: EFβ-scaffold flexibility allows exiting helix reorientation, enabling C-terminal Glu ligand to complete Ca²⁺ coordination.
  • This mechanism explains:
    • Conformational shifts in sensors vs. buffers.
    • Coordination between loop parts and helical motion.
  • Key movement (~2 Å shift) of Glu is critical to driving domain opening.

5. Functional Diversity and Structural Features

  • Different EF-hand proteins utilize various domain opening extents:
    • Calmodulin: ∼40° interhelical change.
    • Calpain: ~18°.
    • Calcyclin: Minimal change.
  • First ligand position affects flexibility:
    • Asp-ligated EF-hands (e.g., Calmodulin, Osteonectin): Large opening needed.
    • Carbonyl-ligated EF-hands (e.g., Calcyclin, Calpain): Simpler pivot, less domain opening.
  • Electrostatic repulsion among negative residues may favor open loop even without Ca²⁺.

6. Other Structural and Functional Insights

  • TnC vs. CaM roles:
    • TnC N-terminal: Specialized Ca²⁺ switch for muscle contraction.
    • CaM: Broad Ca²⁺ sensor, regulating many targets via conformational adaptability.
  • C-terminal domains:
    • TnC: Structural role, stabilizing troponin complex.
    • CaM: Both structural and regulatory, high Ca²⁺ affinity.
  • Helical and linker influences:
    • Exiting helix stability determines response to incomplete Ca²⁺ binding (e.g., Calbindin D9k).
    • Short linkers (CaM, TnC) facilitate coupled motion; longer flexible linkers (S100) allow independent domain shifts.

7. RD’s Take and Learning

  • RD loves the EFβ-scaffold model—a unifying mechanism explaining how Ca²⁺ induces diverse responses.
  • Distinction between sensor vs. buffer proteins is elegantly explained by strain release and conformational opening.
  • The coordination between N- and C-terminal loops, and flexibility of the β-scaffold, are critical in EF-hand biology.
  • RD finds the discussion of ligand identity at the first loop position shaping the opening angle very insightful—relevant for understanding EF-hand adaptability.
  • Concept of paired EF-hands and linker flexibility offers broader context for EF-hand protein engineering or analysis.

Take-home Messages

  • EF-hands are versatile Ca²⁺-binding motifs, using N-terminal flexibility and C-terminal conservation to balance function and stability.
  • EFβ-scaffold model explains two-step Ca²⁺ binding and domain opening.
  • Conformational diversity (e.g., in CaM vs. TnC vs. S100) arises from structural features like loop length, ligand identity, and linker flexibility.
  • Understanding these mechanisms is key to appreciating how EF-hand proteins regulate diverse cellular processes.
  • RD sees this as a foundational paper for understanding Ca²⁺ signaling proteins, blending structural biology with functional insights.