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EF5 Is the High-Affinity Mg(2+) Site in ALG-2.

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The penta-EF-hand protein ALG-2 binds Mg(2+) with high affinity and selectivity in its EF5 domain. This Mg(2+) binding influences protein structure and may affect dimerization, offering insights into other penta-EF-hand proteins.

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Area of Science:

  • Structural biology
  • Biochemistry
  • Molecular biology

Background:

  • The penta-EF-hand (PEF) protein ALG-2 (apoptosis-linked gene 2) is involved in crucial cellular processes like vesicular transport.
  • ALG-2 possesses a metal ion-binding site with high affinity for Mg(2+) and low affinity for Ca(2+).

Purpose of the Study:

  • To elucidate the X-ray structure of Mg(2+)-bound ALG-2des23(wt).
  • To investigate the role of specific residues in Mg(2+) binding and its impact on protein structure and function.

Main Methods:

  • X-ray crystallography to determine the structure of Mg(2+)-bound ALG-2.
  • Site-directed mutagenesis (D169A) to assess the importance of ligand residues.
  • Isothermal titration calorimetry to quantify Mg(2+) binding affinities.

Main Results:

  • The X-ray structure of Mg(2+)-bound ALG-2des23(wt) reveals a C-terminal helix orientation distinct from the Ca(2+)-free form.
  • Mutation of D169 eliminates high-affinity Mg(2+) binding and affects other binding sites.
  • The EF5 domain exhibits an ideal Mg(2+) octahedral coordination, suggesting a high-affinity, selective binding site.

Conclusions:

  • The EF5 domain of ALG-2 provides a high-affinity, selective Mg(2+)-binding site.
  • Mg(2+) binding to ALG-2 can influence protein dimerization and structural conformation.
  • Other PEF proteins, like calpain subunits, may also feature high-affinity Mg(2+)-binding sites.