Two proton translocation pathways in a secondary active multidrug transporter

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biology

Background:

• LmrP is a secondary active multidrug transporter from Lactococcus lactis, belonging to the major facilitator superfamily.
• It utilizes the proton gradient to export lipophilic cations, with ethidium export involving antiport with protons.
• The precise proton translocation mechanism and pathway(s) remained unclear.

Purpose of the Study:

• To elucidate the proton translocation pathways involved in ethidium export by LmrP.
• To investigate the roles of specific residues in proton conduction and substrate interaction.

Main Methods:

• Construction of a 3-D homology model of LmrP based on the GlpT structure.
• Site-directed mutagenesis of key residues (D142, D235, E327) within the predicted internal cavity.
• Analysis of mutant LmrP proteins to assess their function in ethidium translocation.

Main Results:

• The homology model predicted an internal cavity with two clusters of polar residues involved in proton shuttling.
• Mutagenesis revealed that D142 is part of a dedicated proton pathway for ethidium translocation.
• D235 and E327 form an independent pathway, with D235 interacting with protons and E327 modulating D235's pKa and interacting with ethidium.

Conclusions:

• LmrP employs two distinct proton conduction pathways for substrate translocation.
• These findings support a model where major facilitator superfamily proteins have separate domains for substrate binding and ion coupling.
• The study provides detailed insights into the molecular mechanism of proton-assisted drug extrusion by LmrP.