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

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.