Probing structure-function relationships and gating mechanisms in the CorA Mg2+ transport system

Area of Science:

• Structural Biology
• Biophysics
• Molecular Biology

Background:

• CorA Mg(2+) transport proteins feature long ion pores with proposed gating mechanisms at intracellular and periplasmic ends.
• Understanding ion channel and transporter gating is crucial for molecular mechanism elucidation.

Purpose of the Study:

• To probe structure-function relationships in Thermotoga maritima CorA (TmCorA) using a structure-guided engineering approach.
• To investigate the gating mechanisms of the TmCorA Mg(2+) transporter.

Main Methods:

• Structure-guided protein engineering of TmCorA.
• Site-directed mutagenesis to alter pore-lining helices and loops.
• Functional assays to assess Mg(2+) transport and selectivity.

Main Results:

• The intracellular funnel domain acts as an allosteric regulatory module, engineerable for activation or closure.
• A periplasmic gate, involving a proline-induced kink, was identified, with helix-straightening mutations causing gain-of-function.
• Mutations at the narrowest pore constriction confirmed a hydrophobic gate essential for Mg(2+) flux.
• Conserved acidic residues in the periplasmic loop are not critical for function but may aid folding.

Conclusions:

• TmCorA gating involves both allosteric regulation and specific gate structures, including a hydrophobic barrier.
• The findings extend the gating model for the CorA-Alr1-Mrs2 superfamily.
• Identified gating features have broader implications for understanding diverse ion channel and transporter families.