Abstract
The general mechanism of bacterial mechanosensitive channels (MS) has been characterized by extensive studies on a small conductance channel MscS from Escherichia coli (E. coli). However, recent structural studies on the same channel have revealed controversial roles of various channel-bound lipids in channel gating. To better understand bacterial MscS-like channels, it is necessary to characterize homologs other than MscS. Here, we describe the structure of YnaI, one of the closest MscS homologs in E. coli, in its non-conducting state at 3.3 Å resolution determined by cryo electron microscopy. Our structure revealed the intact membrane sensor paddle domain in YnaI, which was stabilized by functionally important residues H43, Q46, Y50 and K93. In the pockets between sensor paddles, there were clear lipid densities that interact strongly with residues Q100 and R120. These lipids were a mixture of natural lipids but may be enriched in cardiolipin and phosphatidylserine. In addition, residues along the ion-conducting pathway and responsible for the heptameric assembly were discussed. Together with biochemical experiments and mutagenesis studies, our results provide strong support for the idea that the pocket lipids are functionally important for mechanosensitive channels.
Highlights
The general mechanism of bacterial mechanosensitive channels (MS) has been characterized by extensive studies on a small conductance channel MscS from Escherichia coli (E. coli)
To shed light on this, we have determined the highresolution structure of YnaI in the closed state at 3.3 Å resolution by cryo electron microscopy. With this greatly improved structure, it becomes clear that the five transmembrane helices (TMs) of YnaI form extended sensor paddles with pockets in between, which are comparably larger than their counterparts in MscS that has only three TMs
The results show a homogenous single peak for the heptameric YnaI on gel filtration chromatography and one clear band on SDS-PAGE (Fig. S1)
Summary
The general mechanism of bacterial mechanosensitive channels (MS) has been characterized by extensive studies on a small conductance channel MscS from Escherichia coli (E. coli). Our structure revealed the intact membrane sensor paddle domain in YnaI, which was stabilized by functionally important residues H43, Q46, Y50 and K93. To shed light on this, we have determined the highresolution structure of YnaI in the closed state at 3.3 Å resolution by cryo electron microscopy (cryo-EM) With this greatly improved structure, it becomes clear that the five TMs of YnaI form extended sensor paddles with pockets in between, which are comparably larger than their counterparts in MscS that has only three TMs. In addition, there are clear lipid densities within those pockets, which have been shown critical for mechanosensing in MscS6. We have performed biochemical analysis on the freshly revealed lipid-binding residues in the YnaI pockets, so to provide valuable insights into the argument about general mechanosensing mechanisms of MscS-like channels
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