Elucidating a role for the cytoplasmic domain in the Mycobacterium tuberculosis mechanosensitive channel of large conductance

Nadia Herrera,Elizabeth S Haswell,Grigory Maksaev,Douglas C Rees

doi:10.1038/s41598-018-32536-6

Abstract

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.

Highlights

Mechanosensitive (MS) channels transduce mechanical stimuli into a variety of cellular responses
These conclusions are supported by FRET observations[23], and models for the open state have been developed through modeling and molecular dynamics simulations[24,25]
The major crystal contacts were mediated by the head-to-head association of two pentamers through the periplasmic loops such that five-fold axes were coincident; similar contacts were observed in the full-length MtMscL structure (Fig. 3A)

Summary

Introduction

Mechanosensitive (MS) channels transduce mechanical stimuli into a variety of cellular responses. 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[6,7,8], 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 kDa4,7,10,11. (i) A proposed role for the C-terminal domain in defining the MscL oligomeric state was motivated by the observation that a C-terminal truncation of SaMscL (SaMscLΔC) formed a tetramer (Fig. 2D)[19]. The available data on the in vivo oligomeric state are consistent with a pentamer for both full length and truncated versions of EcMscL in the membrane[37]

Methods

Results

Conclusion