Quantitating and engineering the ion specificity of an EF-hand-like Ca2+ binding

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biophysics

Background:

• The Escherichia coli D-galactose and D-glucose receptor contains a Ca2+ binding site structurally analogous to eukaryotic EF-hand proteins.
• This bacterial Ca2+ binding site utilizes a pentagonal bipyramidal array of seven oxygen atoms for ion coordination.

Purpose of the Study:

• To investigate the mechanisms governing ion specificity in the bacterial Ca2+ binding site.
• To engineer the Ca2+ binding site to alter its cation selectivity by modifying cavity size and charge.

Main Methods:

• Site-directed mutagenesis was used to replace glutamine 142 with asparagine, glutamate, or aspartate, altering the cavity size and charge.
• Dissociation constants were measured for various metal ions (groups Ia, IIa, IIIa, and lanthanides) to quantify ion selectivity of the engineered sites.

Main Results:

• Engineered sites with smaller side chains (Asn, Asp) bound larger cations up to 50-fold more tightly than the native site.
• Sites with engineered negative charges (Glu, Asp) showed preferences for trivalent over divalent cations, up to 1900-fold higher than the native site.
• Cavity size and negative charge of the coordination array are critical for selective Ca2+ binding.

Conclusions:

• The bacterial Ca2+ binding site's ion selectivity can be modulated by altering the coordination array's properties.
• Engineering the coordination array allows for preferential binding of cations other than Ca2+.
• This study provides insights into the principles of cation selectivity in biological metal-binding sites.