Elucidating a role for the cytoplasmic domain in the Mycobacterium tuberculosis mechanosensitive channel of large conductance (original) (raw)

Microbial survival in dynamic environments requires the ability to successfully respond to abrupt changes in osmolarity. The mechanosensitive channel of large conductance (MscL) is a ubiquitous channel that facilitates the survival of bacteria and archaea under severe osmotic downshock conditions by relieving excess turgor pressure in response to increased membrane tension. A prominent structural feature of MscL, the cytoplasmic C-terminal domain, has been suggested to influence channel assembly and function. In this report, we describe the X-ray crystal structure and electrophysiological properties of a C-terminal domain truncation of the Mycobacterium tuberculosis MscL (MtMscLΔC). A crystal structure of MtMscLΔC solubilized in the detergent n-dodecyl-β-D-maltopyranoside reveals the pentameric, closed state-like architecture for the membrane spanning region observed in the previously solved full-length MtMscL. Electrophysiological characterization demonstrates that MtMscLΔC retains mechanosensitivity, but with conductance and tension sensitivity more closely resembling full length EcMscL than MtMscL. This study establishes that the C-terminal domain of MtMscL is not required for oligomerization of the full-length channel, but rather influences the tension sensitivity and conductance properties of the channel. The collective picture that emerges from these data is that each MscL channel structure has characteristic features, highlighting the importance of studying multiple homologs. Mechanosensitive (MS) channels transduce mechanical stimuli into a variety of cellular responses. MS channels are widely distributed through all kingdoms of life, including bacteria and archaea, where they play a prominent role in maintaining proper osmoregulation 1-5. Sudden changes in osmolarity, such as osmotic downshock during exposure to hypotonic conditions, increases turgor pressure across the cell membrane. This in turn increases the tension within the lipid bilayer and activates a network of MS channels which respond by opening typically nonselective pores in the membrane to release cell contents and alleviate the pressure 1,5-7. Bacterial MS channels are characterized by their relatively high and non-selective conductances, ranging from ~0.1 nS to ~3 nS under defined experimental conditions 6-8 , which is up to several orders of magnitude greater than typical for ion-selective channels 3,6,9. Of these channels, the mechanosensitive channel of large conductance (MscL) exhibits the largest conductance, with an estimated open state pore diameter of ~25-35 Å-large enough for the passage of molecules up to 9 kDa 4,7,10,11. In view of the high conductance yet small subunit size of MscL (Escherichia coli MscL (EcMscL) has 136 residues; Fig. 1), the oligomeric nature of this channel was appreciated from its initial discovery 11. Considerable efforts have been made to establish the molecular mechanism of MscL gating 3,12 , employing a variety of techniques including electrophysiology, biochemical and genetic studies, molecular dynamics