Global structural changes of an ion channel during its gating are followed by ion mobility mass spectrometry

Abstract

Mechanosensitive ion channels are sensors probing membrane tension in all species; despite their importance and vital role in many cell functions, their gating mechanism remains to be elucidated. Here, we determined the conditions for releasing intact mechanosensitive channel of large conductance (MscL) proteins from their detergents in the gas phase using native ion mobilitymass spectrometry (IM-MS). By using IM-MS, we could detect the native mass of MscL from Escherichia coli, determine various global structural changes during its gating by measuring the rotationally averaged collision cross-sections, and show that it can function in the absence of a lipid bilayer. We could detect global conformational changes during MscL gating as small as 3%. Our findings will allow studying native structure of many other membrane proteins.ion mobility mass spectrometry | MscL | membrane proteins | structure function | ion channel gating O ne of the best candidates to explore the gating of mechanosensitive channels is the mechanosensitive channel of large conductance (MscL) from Escherichia coli. The crystal structure of MscL in its closed/nearly closed state from Mycobacterium tuberculosis revealed this channel as a homopentamer (1). Each subunit has a cytoplasmic N-and C-terminal domain as well as two α-helical transmembrane (TM) domains, TM1 and TM2, which are connected by a periplasmic loop. The five TM1 helices form the pore and the more peripheral TM2 helices interact with the lipid bilayer.MscL detects changes in membrane tension invoked by a hypoosmotic shock and couples the tension sensing directly to large conformational changes (1, 2). On the basis of a large body of structural and theoretical data, numerous gating models of MscL have been proposed (3-9). These models agree upon (i) the hydrophobic pore constriction of the channel and (ii) the channel opens by an iris-like rotation-i.e., a tilting and outward movement of transmembrane helices that make the channel wider and shorter (5). This mechanism is supported by patchclamp (10), disulfide cross-linking (11), FRET spectroscopy (12), and site-directed spin labeling EPR experiments (6, 7), as well as computational studies (13-15). So far, direct experimental results have only been observed for short-range local structural changes, and no measure of the overall global structural changes during channel gating have been reported. Because there is no crystal structure available for the open MscL channel, elucidating overall global structural changes from the onset of channel activation is of utmost importance for our understanding of the gating mechanism of mechanosensitive channels. Here, we provide direct experimental evidence for the key areal changes occurring during channel gating by combining our ability to activate MscL in a controlled manner to different subopen states (16) with a native ion mobility-mass spectrometry (IM-MS) approach. Results and DiscussionDetergents that are Unstable in the Gas Phase Allow Detection of MscL in Its Native Form. Native mass ...

Mechanical coupling of the multiple structural elements of the large-conductance mechanosensitive channel during expansion

“…This model was verified and revised by further studies through electron paramagnetic resonance spectroscopy (24) and an electrostatic repulsion test (25). More recently, a study through the native ion mobility-mass spectrometry demonstrated that MscL has the inherent structural flexibility to achieve large global structural changes in the absence of a lipid bilayer (26).…”

Mass spectrometry-enabled structural biology of membrane proteins

“…Recently there has been an upsurge in the use of other non-ionic detergents for native MS analysis of MPs, for example, Triton X-100, tetraethylene glycol monooctyl ether (C8E4), octaethylene glycol monododecyl ether (C12E8), lauryldimethylamine N-oxide (LDAO), and n-octyl-b-D-glucoside (b-OG) (Fig. 4) as it has been shown that these detergent micelles dissociate at much lower activation energies, often resulting in MP ions that are more native-like [95][96][97]. These detergent micelles may be dissociated more easily than those formed by DDM as their non-ionic nature means that the only stabilizing forces are hydrophobic interactions which are weakened in the gas-phase, whilst DDM and b-OG micelles are also stabilized by hydrogenbonding [95].…”

“…4) as it has been shown that these detergent micelles dissociate at much lower activation energies, often resulting in MP ions that are more native-like [95][96][97]. These detergent micelles may be dissociated more easily than those formed by DDM as their non-ionic nature means that the only stabilizing forces are hydrophobic interactions which are weakened in the gas-phase, whilst DDM and b-OG micelles are also stabilized by hydrogenbonding [95]. It has also been proposed that the relative ease of detergent removal may relate to the stability of the protein within Fig.…”

“…Detergents are a poor mimic of the native bilayer, may not permit the retention of MP-lipid interactions [101], and in many cases disturb the correct, functional oligomeric state of MP assemblies [90,95,96]. Consequently, a variety of detergent-free methods have been developed to solubilize MPs for biophysical analyses, namely nanodiscs [102], bicelles [103], liposomes [104], styrene maleic acid lipid particles (SMALPs) (also called lipodisq) [93] and amphipols (Fig.…”

Structural Dynamics of the MSCL C-Terminal Domain

“…Later on, an improved open E. coli MscL (EcMscL) 3D structure was determined using both ensemble and single molecule site-directed fluorofore labelling (SDFL) FRET spectroscopy in combination with MD simulations 6,10,11 in agreement with the X-ray structure of an expanded form of an archaeal MscL homolog from Methanosarcina acetivorans 12 . While the overall gating-related structural changes in MscL have largely been established 8,11,13,14 the structural dynamics and physiological role of the C-terminal domain has thus far been controversial. Several studies have suggested that the C-terminus should remain intact during gating and could function as a molecular sieve 15,16 , while others suggested that this helical bundle should actively participate in gating and that opening of the MscL channel was accompanied by complete dissociation of the bundle 13,17 .…”