Molecular Mechanisms of Mechanosensitive Channel Gating

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 DESCRIPTION (provided by applicant): One reason to study the bacterial mechanosensitive channel of large conductance, MscL, is that it has, and will continue to serve as a molecular paradigm for the investigation of mechanosensory transduction. With a crystal structure of what appears to be a 'nearly-closed' state of M. tuberculosis MscL, the channel has advanced the field considerably by allowing researchers to overlay genetic and molecular analyses, coupled with electrophysiology, onto a structural model. Thus, MscL from E. coli (Ec-MscL), which was the first definitive mechanosensitive channel identified, continues to serve as a tractable model for determining principles for how a protein senses and responds to membrane tension. However, another emerging reason to study MscL is that the channel serves a vital function in maintaining osmotic homeostasis of microbes. It normally serves as a biological emergency release valve; upon osmotic downshock it opens a huge 30Å pore that allows for the rapid release of many accumulated cytoplasmic components, including potassium and glutamate, thus preventing cell lysis. When the channel gates inappropriately it can lead to the death of the microbial cell; it thus is a viable pharmacological target for potential antibiotics. Historically,one of the limitations in the study of MscL function and pharmacology has been the total lack of small molecular probes that bind and modulate the channel. From a High Throughput Screening (HTS) facility on campus, we have identified 18 novel chemical compounds that inhibit the growth of E. coli in a MscL-dependent manner; surprisingly, an additional hit was streptomycin, which, although not its primary mechanism of action, appears to directly bind to and increase the probability of opening the MscL channel; streptomycin also appears to use MscL as a passageway into the cells cytoplasm. In studying streptomycin-MscL interactions, we have developed and refined assays for studying MscL-ligand interactions. These include (1) determining the minimal inhibitory concentrations (MIC) of compounds in the presence and absence of expressed Ec-MscL, MscL orthologues, or the unrelated bacterial mechanosensitive channel MscS as a negative control, (2) measuring the ability of a compound to induce fluxes of potassium and glutamate from the cell in vivo, (3) predicting the compound binding site and how it modifies the protein by using molecular dynamic simulations, and (4) testing these predictions by biochemical and mutagenic means. We will use this array of assays to determine how two additional and very promising compounds identified in the HTS bind to MscL and modulate its activity. These studies will yield insight into mechanosensitive channel gating mechanisms; in addition, co-crystallization of MscL with one or more of these compounds may yield an open state structure for MscL, and the findings could eventually lead to a new generation of antibiotics.