A structured loop modulates coupling between the substrate-binding and dimerization domains in the multidrug...

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biology

Background:

• Secondary active transporters, including the small multidrug resistance (SMR) family, are crucial for moving substrates across cell membranes.
• SMR proteins, like EmrE, are small, dimeric transporters with four transmembrane domains, but the mechanism coupling the dimerization domain (TM4) to the substrate-binding site (TM1-3) remains unclear.

Purpose of the Study:

• To investigate the structure-function relationship of the loop connecting TM3 and TM4 in the SMR protein EmrE.
• To elucidate how this loop mediates conformational changes essential for ion-coupled drug efflux.

Main Methods:

• Solid-state NMR spectroscopy was employed to determine the secondary structure of the EmrE loop in lipid bilayers.
• Structure-function analyses were performed using EmrE mutants in resistance assays in Escherichia coli.

Main Results:

• Solid-state NMR revealed a β-strand secondary structure for the loop connecting TM3 and TM4.
• Mutational analysis showed that both the length and specific residue composition of this loop are critical for EmrE function.
• Altering the loop length or replacing a key hydrophobic residue with glycine abolished ethidium resistance.

Conclusions:

• The loop connecting TM3 and TM4 in EmrE is a structured element essential for ion-coupled transport.
• This structured linker likely plays a conserved role across the SMR family in mediating the conformational switches required for drug efflux.
• Loops in transport proteins can be critical functional components, not just passive linkers.