HapR is designated as a high cell density quorum sensing master regulatory protein of Vibrio cholerae. It is a member of the TetR family protein and functions both as an activator and a repressor by directly communicating with cognate promoters, thus controlling the expression of a plethora of genes in a density-dependent manner. Molecular insights reveal the domain architecture and further unveil the significance of a cross talk between the DNA binding domain and the dimerization domain for the functionality of the wild-type protein. The DNA binding domain is made up of three α-helices, where a helix-turn-helix motif spans between the helices α2 and α3. The essentiality of the glycine-rich linker linking helices α1 and α2 came into prominence while unraveling the molecular basis of a natural non-functional variant of HapR. Subsequently, the importance of linker length was demonstrated. The present study, involving a series of biochemical analyses coupled with molecular dynamics simulation, has illustrated the indispensability of a critical arginine within the linker at position 37 contributing to HapR–DNA binding activity.

Lipids are integral to ion channel function yet delineating mechanisms by which they affect function remains challenging. Within the K _2P family of leak potassium channels, observation of tubular densities interpreted as alkyl chains occupying lateral fenestrations linking the pore and bilayer raised the possibility that lipid access from the bilayer acts as a regulatory mechanism. Here, we present cryo-electron microscopy (cryo-EM) structures of the human leak potassium channel K _2P 6.1 (TWIK2) and mutants in nanodisc and detergent environments that reveal an unusual conformation in the first selectivity filter (SF1) and a pair of two-chain lipids within the channel cavity (denoted the ‘lipid plug’). The chains of each plug lipid occupy separate binding sites that laterally extend to the bilayer from the channel cavity. One, the upper tail, matches the previously identified alkyl chain binding site. The second, the lower tail, occupies a fenestration common with K _2P 1.1 (TWIK1). Together, they demonstrate a two-tailed means to coordinate each plug lipid that offers a reinterpretation of previous observations. Structures of a K _2P 6.1 (TWIK2) mutant that directs the channel to the plasma membrane and an R257A mutant that increases function yield plugged and unplugged forms. Notably, the R257A plugged form shows a change in lipid plug position, indicating a key role for this residue in lipid binding. Molecular dynamics simulations support the stability of the lipid plug and highlight the role of the Arg257 site in plug coordination. Together, our data suggest a model in which occupation of the central cavity by the lipid plug serves as a mechanism to render the TWIK channels inactive and points to the importance of lipid plug removal to create an ion permeable pore. Such a mechanism could provide a potent way for limiting the leak function of K _2P s based on cellular location or other contextual factors.