The structure of an open form of an E. coli mechanosensitive channel at 3.45 A resolution

Abstract

How ion channels are gated to regulate ion flux in and out of cells is the subject of intense interest. The Escherichia coli mechanosensitive channel, MscS, opens to allow rapid ion efflux, relieving the turgor pressure that would otherwise destroy the cell. We present a 3.45 angstrom-resolution structure for the MscS channel in an open conformation. This structure has a pore diameter of approximately 13 angstroms created by substantial rotational rearrangement of the three transmembrane helices. The structure suggests a molecular mechanism that underlies MscS gating and its decay of conductivity during prolonged activation. Support for this mechanism is provided by single-channel analysis of mutants with altered gating characteristics.

Fig. 1

Conformation changes between the A106V and wild type structures (a) The structures are orientated such that the seven fold axis is parallel to the membrane normal. For the A106V structure, one monomer (subunit A) is colored orange, the remaining six subunits gold. The site of the A106V mutation is highlighted. For the wild type structure the A subunit is colored purple, the remaining six subunits B to G are colored lilac. One of the seven portals through which ions are presumed to enter from the cytoplasm is marked. The cytoplasmic domain is essentially unchanged from the wild type structure, there are however profound changes in the transmembrane helices, which are boxed. An orthogonal view is shown in Fig. S3. The environments of A106 and 106V in their respective structures are shown in more detail in Fig. S2. (b) An expanded view of the boxed transmembrane helices of the A subunit. The helices are colored and oriented as above. The blue arrow represents the motion from the closed to the open state. The helices are labeled TM1, 2, 3a and 3b according to the text. In the A106V structure, TM1 and TM2 have undergone a rigid body clockwise rotation relative to their position in the closed structure (labeled TM1^cl and TM2^cl). TM3a also adjusts its position relative to the closed form (TM3a^cl). There is only a small change in TM3b. G113 the pivot point is labeled. (c) The central pore showing only TM3a colored and oriented as Fig. 1a. Comparing the wild type and A106V channel structures, TM3a has pivoted around G113. The blue arrow denotes the movement from closed to open states. As a result of this motion, the helical axis of TM3a in the A106V structure is parallel to the membrane normal, rather than as in the wild type structure at an angle.

Fig. 1

Conformation changes between the A106V and wild type structures (a) The structures are orientated such that the seven fold axis is parallel to the membrane normal. For the A106V structure, one monomer (subunit A) is colored orange, the remaining six subunits gold. The site of the A106V mutation is highlighted. For the wild type structure the A subunit is colored purple, the remaining six subunits B to G are colored lilac. One of the seven portals through which ions are presumed to enter from the cytoplasm is marked. The cytoplasmic domain is essentially unchanged from the wild type structure, there are however profound changes in the transmembrane helices, which are boxed. An orthogonal view is shown in Fig. S3. The environments of A106 and 106V in their respective structures are shown in more detail in Fig. S2. (b) An expanded view of the boxed transmembrane helices of the A subunit. The helices are colored and oriented as above. The blue arrow represents the motion from the closed to the open state. The helices are labeled TM1, 2, 3a and 3b according to the text. In the A106V structure, TM1 and TM2 have undergone a rigid body clockwise rotation relative to their position in the closed structure (labeled TM1^cl and TM2^cl). TM3a also adjusts its position relative to the closed form (TM3a^cl). There is only a small change in TM3b. G113 the pivot point is labeled. (c) The central pore showing only TM3a colored and oriented as Fig. 1a. Comparing the wild type and A106V channel structures, TM3a has pivoted around G113. The blue arrow denotes the movement from closed to open states. As a result of this motion, the helical axis of TM3a in the A106V structure is parallel to the membrane normal, rather than as in the wild type structure at an angle.

Fig. 2

A106V represents an open structure. (a) Superposition of the helical pore (helix TM3a) in MscS. The color scheme is as Fig. 1a. The subunits are labeled; the labels for those in the closed structure are on the inside. The TM3a helices in the A106V MscS structure move out from the centre and align parallel to the 7 fold axis. The blue arrows denote the motion from the closed to the open state. The helical pore is oriented such that one is looking from the periplasm into the cytoplasm. (b) The L105 and L109 side chains, which fill the central pore in closed structure, have moved out of the pore in the A106V structure, creating the open channel by breaking the vapor lock. This motion is akin to the opening of camera iris. The color scheme is the same as Fig. 1a and the orientation as Fig. 2a. (c) The pore formed by the seven TM3a helices in the A106V MscS (left) has a diameter of around 13 Å and is assigned as an open (conducting) state of MscS. The wild type channel has a diameter of 4.8 Å (right) and is thought to represent a closed (non-conducting) channel. The TM3a helices are shown in space filling representation with the same color scheme as Fig. 1a and the same orientation as Fig. 2a. Only residues T93 to N117 are shown. (d) The TM3a helices in the conducting A106V structure are not tightly packed (left), these “spaces” are not wide enough to allow molecules passage but may interact with the lipids. In the non-conducting structure the TM3a helices are tightly packed (right). The open and closed structures are colored and oriented as Fig. 1a. 106V is shown in green for the open structure and A106 in green for the closed structure. Only residues T93 to N117 are shown.

Fig. 2

A106V represents an open structure. (a) Superposition of the helical pore (helix TM3a) in MscS. The color scheme is as Fig. 1a. The subunits are labeled; the labels for those in the closed structure are on the inside. The TM3a helices in the A106V MscS structure move out from the centre and align parallel to the 7 fold axis. The blue arrows denote the motion from the closed to the open state. The helical pore is oriented such that one is looking from the periplasm into the cytoplasm. (b) The L105 and L109 side chains, which fill the central pore in closed structure, have moved out of the pore in the A106V structure, creating the open channel by breaking the vapor lock. This motion is akin to the opening of camera iris. The color scheme is the same as Fig. 1a and the orientation as Fig. 2a. (c) The pore formed by the seven TM3a helices in the A106V MscS (left) has a diameter of around 13 Å and is assigned as an open (conducting) state of MscS. The wild type channel has a diameter of 4.8 Å (right) and is thought to represent a closed (non-conducting) channel. The TM3a helices are shown in space filling representation with the same color scheme as Fig. 1a and the same orientation as Fig. 2a. Only residues T93 to N117 are shown. (d) The TM3a helices in the conducting A106V structure are not tightly packed (left), these “spaces” are not wide enough to allow molecules passage but may interact with the lipids. In the non-conducting structure the TM3a helices are tightly packed (right). The open and closed structures are colored and oriented as Fig. 1a. 106V is shown in green for the open structure and A106 in green for the closed structure. Only residues T93 to N117 are shown.

Fig. 3

Structural features of the channel transition (a) In the closed structure, the side chain of A110 (shown as red sphere) sits in a pocket on one side of the L115 side chain in TM3b from the neighboring G subunit. In the open A106V structure A110 (shown as a yellow sphere) sits in another pocket on the opposite side of the L115 side chain. These different positions of A110, akin to a switch, define the closed and open states. The remainder of the molecule is colored as Figure 1a. The blue arrow defines the motion from closed to open. (b) The methyl group of A102 from the A subunit must cross the face of TM3a from the G subunit, creating a guide to the opening and closing. The A102G mutant opens and closes more easily than the wild type channel, consistent with the removal of this guide. The double mutant A102G/G104A re-introduces a methyl group recreating the guide and is indeed essentially wild type. The molecules are colored as the same as in Figure 1a.

Fig. 4

Mutations at A110 and L115 affect MscS channel opening. Patch-clamp analysis of excised protoplast membrane patches expressing wild type or mutant MscS protein. (a) Wild type (WT) channels show step-like opening and closing events. Substitution of A110 to glycine produces channels that are unstable in the open state, thus openings are rapidly followed by closures giving channel activity of a flickery appearance. Substitution to valine leads to a channel that does not open fully; its open state is stable but exhibits a decreased conductance. (b) Exchange of L115 for a smaller valine residue disrupts the ability of the channel to open fully and/or remain open. Vertical scale bar, 50 pA; horizontal scale bar, 200 ms.

Fig. 5

Alanine at position 102 is important to maintain the open state of MscS. (a)-(c) Patch clamp recordings of wild type (WT) and A102G mutant MscS channels. (a) A102G mutation renders the channels unstable in the open state. Vertical scale bar, 50 pA; horizontal scale bar, 200 ms. (b) Prolonged application of pressure (shown in lower panel of both traces) causes open wild type (WT) channels to adapt and become non-conducting. Thus the current trace (upper panel in each recording) returns to baseline in the presence of applied pressure. A102G mutant channels rapidly adapt to sustained pressure. Vertical scale bar, 200 pA and 340 mm Hg; horizontal scale bar, 10 s. (c) Reciprocal double mutations at positions 102/101 or 102/104 return the stable fully open conformation. Vertical scale bar, 50 pA; horizontal scale bar, 200 ms. (d) Mutant cycle analysis(25) of the gating pressures (derived from patch clamp analysis) for each single and double mutant. The P_L:P_S ratio for each single and double mutant was measured as described in methods(17). The P_L:P_S ratio difference between the single mutants and wild type, and double mutants and single mutants, was calculated to measure the change associated with the introduction of the indicated mutation. Summation across the diagram (i.e. comparing the effect of introducing a specific mutation into either wild type or mutant channel) indicates that whilst residue G101 does not couple to A102 (difference value of 0.02 is close to zero), G104 and A102 appear to energetically interact (difference value of 0.38 is greater than zero) (25-30)