Comparing and Contrasting Escherichia coliand Mycobacterium tuberculosis Mechanosensitive Channels (MscL)

Abstract

Sequence analysis of 35 putative MscL homologues was used to develop an optimal alignment for Escherichia coli and Mycobacterium tuberculosis MscL and to place these homologues into sequence subfamilies. By using this alignment, previously identified E. coli MscL mutants that displayed severe and very severe gain of function phenotypes were mapped onto the M. The recent crystal structures of two bacterial ion channels, the KcsA potassium channel and the mechanosensitive channel MscL, provide unique opportunities to study ion channel structure-function relationships (1, 2). Concerning the MscL system, recent work has attempted to rationalize the extensive functional studies on Escherichia coli MscL (Eco-MscL) 1 (17). This difference may result from protein structural differences, a difference in interactions with lipids, or both.Sequence alignment is essential to map previously studied E. coli GOF mutations onto the M. tuberculosis MscL sequence. In this work we report an optimal sequence alignment of 35 MscL homologues and an analysis of regions of conservation and variability. Consistent with previous studies, we find greater conservation in the transmembrane regions than in the loop or intracellular regions. Interestingly, the various channels clearly fall into subfamilies based on sequence similarity, with Eco-MscL and Tb-MscL in different subfamilies.By using the optimal alignment, we have prepared Tb-MscL analogues of several critical Eco-MscL GOF mutations (Fig. 1A). Perhaps surprisingly, we find that several well established Eco-MscL GOF mutants do not translate to the Tb-MscL system. We also directly evaluate a predicted intersubunit hydrogen bond in the Tb-MscL crystal structure (Fig. 1B). Crosslinking studies establish that these residues are indeed close in the reconstituted channel and firmly establish the pentameric nature of the channel. Mutations of this pair generally lead to GOF mutants, suggesting an important functional role for this specific region of the channel. Interestingly, no analogous interaction is apparent in the Eco-MscL alignment. Our results indicate that the functional studies performed on the Eco-MscL channel may not map directly onto the Tb-MscL crystal structure. MATERIALS AND METHODSSequence Analysis-Multiple sequence alignments were obtained using alignment of multiple sequences (AMPS) (20,21), and consensus group analysis was performed using multiple EM for motif elicitation (MEME) (22,23). The alignment was broken into regions, extracellular

Lactococcus lactis Uses MscL as Its Principal Mechanosensitive Channel

“…This is where the mechanosensitive channels are thought to play a role by opening in response to the increased membrane tension effected by the rapid increase in cell volume. The best known example of a channel in this role is the mechanosensitive channel of large conductance from Escherichia coli (MscL Ec ), but homologues are present in most eubacteria (5)(6)(7). MscL opens near the lytic tension limit of the bacterial membrane.…”

Gating the bacterial mechanosensitive channel MscL in vivo

“…For MscL, the atomic resolution of its structure (5), computer-assisted modeling (8), biophysical calibration (7), and genetic dissection (6,(11)(12)(13) have made it a premier molecular model to study mechanosensory transduction. But the insights gained in these studies cannot be easily translated into insights in bacterial physiology.…”

Functional Design of Bacterial Mechanosensitive Channels

“…However, the molecular entity(s) responsible for MscS activity remained undefined. Although homologues of MscL are observed throughout the bacterial kingdom (19,20), and in all cases studied have been shown to encode MS channels (20,21), no homologues were found within E. coli that could encode the MscS activity, thus implying it belonged to a new gene family. Recently, Levina et al (22) described a gainof-function (GOF) mutant of the E. coli kefA (also in the data base as aefA) gene, which subsequently led to the discovery of a large family of genes.…”