A Salt Bridge Pre-arranges the Structure of the ABC Transporter BmrA for Proper NBD Dimerization

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biology

Background:

• ATP-binding cassette (ABC) transporters mediate membrane translocation via communication between nucleotide-binding domains (NBDs) and transmembrane domains (TMDs).
• Type IV ABC exporters utilize two coupling helices (CHs) for NBD-TMD communication, but the stabilization mechanism of this interface is unknown.
• The linker region in Bacillus multidrug resistance ATP (BmrA) transporter is positioned near the CHs, suggesting a potential role in interface stabilization.

Purpose of the Study:

• To investigate the role of the linker region in stabilizing the NBD-TMD interface in type IV ABC exporters.
• To identify specific residues and interactions critical for maintaining functional communication within ABC transporters.

Main Methods:

• Alanine mutagenesis of the BmrA linker region to assess the impact on transport and ATPase activity.
• Cryo-electron microscopy (Cryo-EM) to determine the structural consequences of key mutations.
• Further mutagenesis to identify interaction partners for critical linker residues.

Main Results:

• Substitution of Glu326 in the linker region abolished BmrA ATPase activity and impaired transport.
• Cryo-EM analysis of the Glu326Ala mutant revealed a disrupted NBD-TMD arrangement with uncoupled CHs.
• Mutagenesis identified Lys217 in CH2 as a potential interaction partner for Glu326.

Conclusions:

• A conserved Glu326-Lys217 salt bridge stabilizes the CH-NBD interface in BmrA.
• This salt bridge is essential for promoting NBD dimerization and functional coupling in type IV ABC exporters.
• The linker region plays a critical, previously unrecognized role in ABC transporter function, conserved across bacterial and human ABC-C transporters.