No abstract

No abstract

The large conductance mechanosensitive channel (MscL), acts as an osmoprotective emergency valve in bacteria by opening a large, water-filled pore in response to changes in membrane tension. In its closed configuration, the last 36 residues at the C-terminus form a bundle of five α-helices co-linear with the five-fold axis of symmetry. Here, we examined the structural dynamics of the C-terminus of EcMscL using site-directed spin labelling electron paramagnetic resonance (SDSL EPR) spectroscopy. These experiments were complemented with computational modelling including molecular dynamics (MD) simulations and finite element (FE) modelling. Our results show that under physiological conditions, the C-terminus is indeed an α-helical bundle, located near the five-fold symmetry axis of the molecule. Both experiments and computational modelling demonstrate that only the top part of the C-terminal domain (from the residue A110 to E118) dissociates during the channel gating, while the rest of the C-terminus stays assembled. This result is consistent with the view that the C-terminus functions as a molecular sieve and stabilizer of the oligomeric MscL structure as previously suggested.The bacterial mechanosensitive channel of large conductance (MscL), has become a prototype molecular system in the study of mechanotransduction and its evolutionary origins 1,2 . The three-dimensional (3D) structure of MscL from Mycobacterium tuberculosis (MtMscL) in its closed conformation was resolved at 3.5 Å 3 . The channel is a homopentamer consisting of N-and C-terminal domains facing the cytoplasm and TM1 and TM2 transmembrane helices connected by a periplasmic loop [3][4][5][6] . The closed channel structure was also determined in the lipid bilayer by site-directed spin labelling electron paramagnetic resonance (SDSL EPR) spectroscopy 7 . Moreover, the open channel structure was also determined by SDSL EPR spectroscopy 8 allowing for estimation of the size of the open channel pore in a very good agreement with electrophysiological sieving studies 9 . Later on, an improved open E. coli MscL (EcMscL) 3D structure was determined using both ensemble and single molecule site-directed fluorofore labelling (SDFL) FRET spectroscopy in combination with MD simulations 6,10,11 in agreement with the X-ray structure of an expanded form of an archaeal MscL homolog from Methanosarcina acetivorans 12 . While the overall gating-related structural changes in MscL have largely been established 8,11,13,14 the structural dynamics and physiological role of the C-terminal domain has thus far been controversial. Several studies have suggested that the C-terminus should remain intact during gating and could function as a molecular sieve 15,16 , while others suggested that this helical bundle should actively participate in gating and that opening of the MscL channel was accompanied by complete dissociation of the bundle 13,17 . More recently, the X-ray structure of the EcMscL C-terminus domain was obtained at 1. 45 Å 18 , suggesting that even in the absence ...