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Structure of a tetrameric MscL in an expanded intermediate state.

ABSTRACT: The ability of cells to sense and respond to mechanical force underlies diverse processes such as touch and hearing in animals, gravitropism in plants, and bacterial osmoregulation. In bacteria, mechanosensation is mediated by the mechanosensitive channels of large (MscL), small (MscS), potassium-dependent (MscK) and mini (MscM) conductances. These channels act as 'emergency relief valves' protecting bacteria from lysis upon acute osmotic down-shock. Among them, MscL has been intensively studied since the original identification and characterization 15 years ago. MscL is reversibly and directly gated by changes in membrane tension. In the open state, MscL forms a non-selective 3 nS conductance channel which gates at tensions close to the lytic limit of the bacterial membrane. An earlier crystal structure at 3.5 A resolution of a pentameric MscL from Mycobacterium tuberculosis represents a closed-state or non-conducting conformation. MscL has a complex gating behaviour; it exhibits several intermediates between the closed and open states, including one putative non-conductive expanded state and at least three sub-conducting states. Although our understanding of the closed and open states of MscL has been increasing, little is known about the structures of the intermediate states despite their importance in elucidating the complete gating process of MscL. Here we present the crystal structure of a carboxy-terminal truncation mutant (Delta95-120) of MscL from Staphylococcus aureus (SaMscL(CDelta26)) at 3.8 A resolution. Notably, SaMscL(CDelta26) forms a tetrameric channel with both transmembrane helices tilted away from the membrane normal at angles close to that inferred for the open state, probably corresponding to a non-conductive but partially expanded intermediate state.

SUBMITTER: Liu Z

PROVIDER: S-EPMC2737600 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Structure of a tetrameric MscL in an expanded intermediate state.

Liu Zhenfeng Z Gandhi Chris S CS Rees Douglas C DC

Nature 20090823 7260

The ability of cells to sense and respond to mechanical force underlies diverse processes such as touch and hearing in animals, gravitropism in plants, and bacterial osmoregulation. In bacteria, mechanosensation is mediated by the mechanosensitive channels of large (MscL), small (MscS), potassium-dependent (MscK) and mini (MscM) conductances. These channels act as 'emergency relief valves' protecting bacteria from lysis upon acute osmotic down-shock. Among them, MscL has been intensively studied ...[more]

PMID: 19701184

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Project description:The tension-driven gating transition in the large mechanosensitive channel MscL proceeds through detectable states of intermediate conductance. Gain-of-function (GOF) mutants with polar or charged substitutions in the main hydrophobic gate display altered patterns of subconducting states, providing valuable information about gating intermediates. Here we present thermodynamic analysis of several GOF mutants to clarify the nature and position of low-conducting conformations in the transition pathway. Unlike wild-type (WT) MscL, which predominantly occupies the closed and fully open states with very brief substates, the mild V23T GOF mutant frequently visits a multitude of short-lived subconducting states. Severe mutants V23D and G22N open in sequence: closed (C) --> low-conducting substate (S) --> open (O), with the first subtransition occurring at lower tensions. Analyses of equilibrium state occupancies as functions of membrane tension show that the C-->S subtransition in WT MscL is associated with only a minor conductance increment, but the largest in-plane expansion and free energy change. The GOF substitutions strongly affect the first subtransition by reducing area ((Delta)A) and energy ((Delta)E) changes between C and S states commensurably with the severity of mutation. GOF mutants also exhibited a considerably larger (Delta)E associated with the second (S-->O) subtransition, but a (Delta)A similar to WT. The area changes indicate that closed conformations of GOF mutants are physically preexpanded. The tension dependencies of rate constants for channel closure (k(off)) predict different positions of rate-limiting barriers on the energy-area profiles for WT and GOF MscL. The data support the two-gate mechanism in which the first subtransition (C-->S) can be viewed as opening of the central (M1) gate, resulting in an expanded water-filled "leaky" conformation. Strong facilitation of this step by polar GOF substitutions suggests that separation of M1 helices associated with hydration of the pore in WT MscL is the major energetic barrier for opening. Mutants with a stabilized S1 gate demonstrate impeded transitions from low-conducting substates to the fully open state, whereas extensions of S1-M1 linkers result in a much higher probability of reverse O-->S transitions. These data strongly suggest that the bulk of conductance gain in the second subtransition (S-->O) occurs through the opening of the NH2-terminal (S1) gate and the linkers are coupling elements between the M1 and S1 gates.

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Structure of a tetrameric MscL in an expanded intermediate state.

Publications

Structure of a tetrameric MscL in an expanded intermediate state.

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