Water-mediated interactions enable smooth substrate transport in a bacterial efflux pump

Area of Science:

• Microbiology
• Structural Biology
• Computational Biology

Background:

• Gram-negative bacteria utilize Resistance-Nodulation-cell Division (RND) superfamily efflux pumps for multi-drug resistance.
• The Escherichia coli inner-membrane protein AcrB is a key polyspecific transporter in this class.
• A functional rotation mechanism for AcrB has been proposed but lacks mechanistic and energetic validation.

Purpose of the Study:

• To computationally investigate the proposed functional rotation mechanism of the AcrB efflux pump.
• To elucidate the molecular determinants and energetics governing substrate translocation.
• To provide a mechanistic understanding of polyspecific transport in Gram-negative bacteria.

Main Methods:

• Development of a computational protocol to simulate the AcrB functional rotation mechanism.
• Utilizing multi-bias molecular dynamics simulations to track doxorubicin translocation.
• Estimation of the free energy profile associated with substrate transport.

Main Results:

• A molecular mechanism for AcrB function was elucidated, consistent with experimental data.
• Conformational changes in AcrB facilitate the formation of a structured water layer within the transport channel.
• This water layer aids in substrate hydration and smooth diffusion along a favorable free energy profile.

Conclusions:

• A novel molecular mechanism for polyspecific transport involving structured water has been identified.
• Water plays a crucial role by screening strong substrate-protein interactions.
• Findings advance understanding of multi-drug efflux and inform the design of efflux pump inhibitors.