Dihydrostreptomycin Directly Binds to, Modulates, and Passes through the MscL Channel Pore

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Abstract
The primary mechanism of action of the antibiotic dihydrostreptomycin is binding to and modifying the function of the bacterial ribosome, thus leading to decreased and aberrant translation of proteins; however, the routes by which it enters the bacterial cell are largely unknown. The mechanosensitive channel of large conductance, MscL, is found in the vast majority of bacterial species, where it serves as an emergency release valve rescuing the cell from sudden decreases in external osmolarity. While it is known that MscL expression increases the potency of dihydrostreptomycin, it has remained unclear if this effect is due to a direct interaction. Here, we use a combination of genetic screening, MD simulations, and biochemical and mutational approaches to determine if dihydrostreptomycin directly interacts with MscL. Our data strongly suggest that dihydrostreptomycin binds to a specific site on MscL and modifies its conformation, thus allowing the passage of K+ and glutamate out of, and dihydrostreptomycin into, the cell. Streptomycin is one of the original and best studied antibiotics. Its primary mechanism of action is the interference with protein synthesis by binding to and modifying the function of the bacterial ribosome. However, the antibiotic is quite large, bulky, and is charged, so the mechanisms by which it accesses the inside of the bacterial cell have remained largely unknown. Previously, we have found that the expression of a bacterial mechanosensitive channel, MscL, increases the potency of a variant of this antibiotic, dihydrostreptomycin. Here, we define the dihydrostreptomycin binding site on the MscL channel. We also show how this antibiotic modifies the channel and opens the pore, allowing the diffusion of solutes, such as potassium and glutamate, from the cytoplasm of the cell out to the medium. Finally, we provide evidence that dihydrostreptomycin can pass through MscL and that the channel is thus one pathway by which the drug accesses the inside of the bacterial cell.
Funding Information
  • National Institute of General Medical Sciences (GM061028)
  • Welch Foundation (I-1420)
  • Cancer Prevention and Research Institute of Texas (RP100146)
  • American Heart Association (12SDG8740012)
  • National Natural Science Foundation of China (81473254)

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