Structural Dynamics of the MscL C-terminal Domain

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.

Figure 1

3D homology model of the closed EcMscL channel. (A) Single MscL subunit (rose pink) showing amino acid residues comprising the A110-S136 segment of the C-terminal bundle encompassing 27 residues subjected to cysteine scanning mutagenesis (blue spheres). The single MscL monomer is represented as a part of the channel pentamer according to the 3D homology model of EcMscL as described in the methods (left). Linear representation of the membrane topology of EcMscL (right). The N- and C-terminal domains, transmembrane helices TM1 and TM2 and the periplasmic and cytoplasmic loops are represented by rose pink and blue rectangles, respectively. (B) Sequence alignment of the C-terminal bundle of EcMscL and MtMscL. Identical residues are highlighted in black. The numerical equivalence between the residues shown in the figure was generated using a ClustalW pairwise sequence alignment between the two helical bundle sequences.

Figure 2

Residue-specific environmental parameter profiles obtained for the A110–S136 segment of the C-terminal bundle. (A) Mobility parameter ΔH₀ ^–1 indicates the greatest mobility for the first seven residues A110–T116 and three last residues N134–S136, which suggests these residues form a free loop and have no defined secondary structure, respectively (left). A periodicity in the mobility of the spin label from T116 through to E131 residues is consistent with the observed α-helical structure of this portion of the C-terminal bundle (right) in the crystal structure of MtMscL (D108–Q123 in MtMscL), which is not considered to be in the fully open state but an expanded state (Li, Jie, et al., 2015, PNAS). Importantly however, MaMscL has a vastly different C-terminus compared to the EcMscL in terms of the number of helices, sequence and structure. Nevertheless, the transmembrane helices share high similarity in sequence and gating mechanism. (B) NiEdda accessibility parameter ΠNiEdda shows high accessibility of most helical bundle residues to NiEdda and thus to the aqueous compartment consistent with the cytoplasmic location of the bundle (left). The profile of ΠNiEdda is also roughly correlates to the profile of the mobility parameter ΔH₀ ⁻¹ shown in (A). Accessibility to NiEdda indicated in blue on the crystal structure of the helical bundle (right).

Figure 3

Examples of EPR spectra of the C-terminal residues of EcMscL. X-band EPR spectra of underlabelled and fully spin-labelled A110C, E119C and Q132C mutants showing increased spin mobility at the top and the bottom of the A110–S136 segment of the helical bundle and reduced mobility in the middle of the A110–S136 segment. All spectra were obtained using loop-gap resonator with the microwave power of 2 mW and field modulation of 1 G.

Figure 4

Mobility parameter ΔH₀ ⁻¹ profiles of the C-terminal helical bundle. The mobility parameter ΔH₀ ⁻¹ was obtained in the closed (black) and open (red) configuration of MscL. The spin-labelled mutant channels were opened by applying 25 mol% LPC to liposomes reconstituted with the mutant proteins, as previously reported^,. Except for the first ten amino acid residues (A110 through E119) showing higher mobility in the open channel configuration compared to the closed channel, the profiles are almost identical showing no difference in the mobility of the remaining 17 residues (V120 through S136) between the closed and the open MscL channel.

Figure 5

Patch-clamp recording from purified MscL mutant V120C reconstituted into liposomes. (A) The MscL-V120C mutant current recorded upon the application of 10 mmHg suction steps onto the patch area. The channel currents (top) and the negative pressure applied to the inside-out azolectin liposome patch through the patch pipette (bottom) are shown. (B) Activation of the mutant channel by application of 3 μM LPC in the patch pipette, which is much less than 25 mol% used in EPR experiments (Fig. 4). (Note that it took ∼2 min for LPC to diffuse inside the pipette to reach and incorporate in the monounsaturated (18:1) POPC liposome patch and activate the channel.) The channel activity is characterized by brief channel openings. Expanded views show the openings of the MscL-V120C mutant in the presence (left) and absence of suction (right) after LPC incorporation into the liposome patch. (C) The activation of the MscL mutant channel in the presence of 5 μM LPC in the patch pipette before, during and after application of suction to the pipette. Note longer openings of multiple channels when compared to 3 μM LPC recordings. Pipette potential was +30 mV.

Figure 6

Conformations of the MscL C-terminal bundle obtained during MscL gating using all-atom molecular dynamics simulations. EcMscL structure (top panel) showing the helical bundle in the resting (closed) state after the initial relaxation of ∼60 ns. (A) and in the open state (B). The helical bundle is coloured in rose pink. The close-up perspective of the C-terminal bundle (C) at rest, (D) at the open state and (E) the overlay of the two states. Top, middle and bottom interactions between the residues E (red), R (blue), D (red) and S (green). (F) Change in the total interaction energy during the channel gating is plotted against the alpha-carbon to alpha-carbon distance of the residues forming the top, middle and bottom belts. The middle belt residue interaction is electrostatic due to the salt bridge formation between E124 and R126 and D127 residues of the adjacent subunit. A tight and stable hydrogen bond also exists between R135 and S136 of the adjacent subunit (bottom belt). (G) The number of hydrogen bonds in the top belt decreases dramatically from ~ 20 to zero during the channel opening, while the number of hydrogen bonds in the bottom belt remains stable. For all the hydrogen bond calculations, we used a donor-acceptor distance of 3.5 Å and angle cut-off of 30°. Overall, the figure shows low interaction energy in the top belt and high interaction energy in the middle belt. The helical bundle dissociates during MscL opening around the top part but remains intact in the middle and bottom parts during 210 ns simulation time (206 ns of the stretching simulation plus 4 ns of the equilibration step; therefore, 206 + 4 = 210 ns).

Figure 7

C-terminal helical bundle structural dynamics using finite element (FE) simulation for water-salt (KCl) environment. (A) Side and top views of the closed state and (B) open state of MscL are shown. In the open state (where the pore diameter is D ∼ 30 Å and the membrane thins from 35 Å to ~30 Å) there is outward bending in the upper part of the helical bundle compared to the resting (equilibrated) state, while the rest of the bundle does not change during the channel opening.

Figure 8

A diagram showing the main structural conformations during MscL opening. The C-terminal bundle remains largely intact during MscL structural change from the closed to the open state. The model also shows the MscL N-terminal domain (red rectangle) fusing with the TM1 transmembrane helix to form a single continuous α-helix in the fully open channel^,.