On the structure of the N-terminal domain of the MscL channel: helical bundle or membrane interface.
ABSTRACT The mechanosensitive channel of large conductance, MscL, serves as a biological emergency release valve protecting bacteria from acute osmotic downshock and is to date the best characterized mechanosensitive channel. A well-recognized and supported model for Escherichia coli MscL gating proposes that the N-terminal 11 amino acids of this protein form a bundle of amphipathic helices in the closed state that functionally serves as a cytoplasmic second gate. However, a recently reexamined crystal structure of a closed state of the Mycobacterium tuberculosis MscL shows these helices running along the cytoplasmic surface of the membrane. Thus, it is unclear if one structural model is correct or if they both reflect valid closed states. Here, we have systematically reevaluated this region utilizing cysteine-scanning, in vivo functional characterization, in vivo SCAM, electrophysiological studies, and disulfide-trapping experiments. The disulfide-trapping pattern and functional studies do not support the helical bundle and second-gate hypothesis but correlate well with the proposed structure for M. tuberculosis MscL. We propose a functional model that is consistent with the collective data.
- SourceAvailable from: Ankush Saddhe[Show abstract] [Hide abstract]
ABSTRACT: Mechanosensitive channels are membrane proteins that open and shut in response to mechanical forces produced by osmotic pressure, sound, touch and gravity. These channels are involved in multiple physiological functions including hypoosmotic pressure, pain, hearing, blood pressure and cell volume regulation. In plants, these channels play a major role in proprioception, gravity sensing and maintenance of plastid shape and size. In the present study, we identified the mechanosensitive channel of small conductance like (MscS) homologue gene family in rice and analyzed their structure, phylogenetic relationship, localization and expression pattern. Five MscS like genes of rice (OsMSL) were found to be distributed on four chromosomes and clustered into two major groups. Subcellular localization predictions of the OsMSL family revealed their localization to plasma membrane, plastid envelope and mitochondria. The predicted gene structure, bonafide conserved signature motif, domain and the presence of transmembrane regions in each OsMSL strongly supported their identity as members of MscS-like gene family. Furthermore, in silico expression analysis of OsMSL genes revealed differential regulation patterns in tissue specific and abiotic stress libraries. These findings indicate that the in silico approach used here successfully identified in a genome-wide context MscS like gene family in rice, and further suggest the functional importance of MscS-like genes in rice.Plant Gene. 01/2015; 1:8-17.
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ABSTRACT: MscL, the highly conserved bacterial mechanosensitive channel of large conductance, is one of the best studied mechanosensors. It is a homopentameric channel that serves as a biological emergency release valve that prevents cell lysis from acute osmotic stress. We previously showed that the periplasmic region of the protein, particularly a single residue located at the TM1/periplasmic loop interface, F47 of Staphylococcus aureus and I49 of Escherichia coli MscL, plays a major role in both the open dwell time and mechanosensitivity of the channel. Here, we introduced cysteine mutations at these sites and found they formed disulfide bridges that decreased the channel open dwell time. By scanning a likely interacting domain, we also found that these sites could be disulfide trapped by addition of cysteine mutations in other locations within the periplasmic loop of MscL, and this also led to rapid channel kinetics. Together, the data suggest structural rearrangements and protein-protein interactions that occur within this region upon normal gating, and further suggest that locking portions of the channel into a transition state decreases the stability of the open state.Biophysical Journal 01/2014; 106(2):375-81. · 3.83 Impact Factor
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ABSTRACT: The bacterial mechanosensitive channel MscL, a small protein mainly activated by membrane tension, is a central model system to study the transduction of mechanical stimuli into chemical signals. Mutagenic studies suggest that MscL gating strongly depends on both intra-protein and interfacial lipid-protein interactions. However, there is a gap between this detailed chemical information and current mechanical models of MscL gating. Here, we investigate the MscL bilayer-protein interface through molecular dynamics simulations, and take a combined continuum-molecular approach to connect chemistry and mechanics. We quantify the effect of membrane tension on the forces acting on the surface of the channel, and identify interactions that may be critical in the force transduction between the membrane and MscL. We find that the local stress distribution on the protein surface is largely asymmetric, particularly under tension, with the cytoplasmic side showing significantly larger and more localized forces, which pull the protein radially outward. The molecular interactions that mediate this behavior arise from hydrogen bonds between the electronegative oxygens in the lipid headgroup and a cluster of positively charged lysine residues on the amphipathic S1 domain and the C-terminal end of the second trans-membrane helix. We take advantage of this strong interaction (estimated to be 10–13 kT per lipid) to actuate the channel (by applying forces on protein-bound lipids) and explore its sensitivity to the pulling magnitude and direction. We conclude by highlighting the simple motif that confers MscL with strong anchoring to the bilayer, and its presence in various integral membrane proteins including the human mechanosensitive channel K2P1 and bovine rhodopsin. OPEN ACCESSPLoS ONE 12/2014; 9(12):e113947. · 3.53 Impact Factor
On the Structure of the N-Terminal Domain of the MscL Channel: Helical
Bundle or Membrane Interface
Irene Iscla, Robin Wray, and Paul Blount
Department of Physiology, University of Texas, Southwestern Medical Center at Dallas, Dallas, Texas
protecting bacteria from acute osmotic downshock and is to date the best characterized mechanosensitive channel. A well-
recognized and supported model for Escherichia coli MscL gating proposes that the N-terminal 11 amino acids of this protein
reexamined crystal structure of a closed state of the Mycobacterium tuberculosis MscL shows these helices running along the
cytoplasmic surface of the membrane. Thus, it isunclear if one structural modelis correct or if theyboth reflectvalid closedstates.
electrophysiological studies, and disulfide-trapping experiments. The disulfide-trapping pattern and functional studies do not
propose a functional model that is consistent with the collective data.
The mechanosensitive channel of large conductance, MscL, serves as a biological emergency release valve
Mechanosensation is essential for all forms of life, underly-
ing many vital processes such as osmoregulation, gravi-
tropism, the senses of hearing, balance, and touch as well as
heart mechanoelectric feedback and blood pressure regula-
tion (1–3). The transducers involved in these processes are
often mechanosensitive channels. The molecular entities of
most of the mechano-transducers in higher organisms have
not yet been identified; in contrast, several mechanosensitive
(MS) channels inbacteria have beencloned,and thestructure
of one representative for each of the two major families of
these microbial channels has been resolved by x-ray crys-
Four MS channel activities have been characterized in
Escherichia coli: the mechanosensitive channel of large
conductance, MscL; smaller conductance, MscS; minicon-
ductance, MscM; and one that is K1-regulated, MscK (6–9).
BothMscLand MscSchannelsactascoordinated emergency
valves allowing rapid solute release and homeostatic adjust-
lower-osmolarity environment). Indeed, the double mutant
DmscL/DmscS bacterial strain (but not the single mutants)
shows more than a 10-fold increase in cell lyses on osmotic
downshock compared with the parental strain (7).
MscL from E. coli was the first cloned (10) and is to date
the best understood MS channel from any species (11). It is a
relatively small protein of 136 amino acids. The crystal
structure of the ortholog from Mycobacterium tuberculosis
(Tb-MscL) was solved to 3.5 A˚resolution (5) and showed
that the channel is a homopentamer in which each subunit
contains two transmembrane domains (TMDs); both the
N- and C-terminal regions are cytoplasmic (12). The most
N-terminal region of the channel was not resolved in the first
published structure for Tb-MscL (5). Subsequently, based on
this structure and disulfide-trapping experiments, a detailed
model for the gating of E. coli MscL (Eco-MscL) was pre-
dicted (13,14). Because the work was performed in the lab-
oratories of Sukharev and Guy, this model has been referred
to as the Sukharev-Guy or SG model; for simplicity we use
the latter. The SG model proposed that the extreme N-ter-
minal region (named S1) forms a bundled helix when the
channel is in a closed state. This bundle serves as a second
gate, remaining closed until most of the expansion of the
channel has already occurred (Fig. 1) (13,14). However, in a
newly revised version of the crystal structure for Tb-MscL,
the S1 region was better resolved and modeled, thus showing
S1 to have a helical structure running parallel to the cyto-
plasmic membrane (15) instead of forming the tight bundle
proposed by the SG model (Fig. 1).
Here we present a systematic study of the S1 domain of
E.coli MscL to determine its role in channel gating in lightof
these twoexistingmodels. Wehavescanned theentire region
with cysteines and performed functional assays, an in vivo
SCAM, electrophysiological characterization, and in vivo
disulfide-trapping assays. Our results correlate well with the
predicted structure of the S1 domain by the Tb-MscL crystal,
counter predictions made by the SG model, and suggest an
alternative role for gating.
MATERIALS AND METHODS
Strains and cell growth
The cysteine mutant library was generated using the Mega Primer method as
described previously (16). Mutants were inserted within the pB10d expression
Submitted December 10, 2007, and accepted for publication May 12, 2008.
Address reprint requests to Paul Blount, Department of Physiology, Univer-
sity of Texas, Southwestern Medical Center at Dallas, 5323 Harry Hines
Blvd., Dallas, TX 75390-9040. Tel.: 214-645-6014; Fax: 214-645-6019;
Editor: Richard W. Aldrich.
? 2008 by the Biophysical Society
Biophysical JournalVolume 95 September 20082283–2291 2283
construct, a modified pB10b plasmid (17–19), in which a methylation site,
overlapping with the Xba consensus site, was changed (TCTAGAT to
TCTAGAG). E. coli strain PB104 (DmscLTCm) (17) was used for the in
vivo cysteine-trapping experiments and for electrophysiological analysis
in which MscS was used as an internal control. The E. coli FRAG-1 deriva-
tive strain, MJF455 DmscLTCm, DmscS (7), was used for viability experi-
ments after osmotic downshock and in vivo SCAM experiments. MTSET
(2-(trimethylammonium)ethyl methanethiosulfonate bromide) and MTSES
(2-sulfonatoethyl methanethiosulfonate sodium salt) were obtained from
Toronto Research Chemicals (North York, ON, Canada). Cultures were
routinely grown in Lennox Broth medium (LB) (Fisher Scientific, Pittsburgh,
incubator at 37?C and rotated at 250 cycles/min. Expression was induced by
addition of 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG) (Anatrace,
Western blot analysis
Western blots were performed as described (21). Briefly, colonies were
supplemented with 1 M NaCl and 2 mM IPTG. When grown to an OD600of
0.3–0.4, the cultures were diluted 20-fold in prewarmed (37?C) distilled
shaker-incubator for 20 min. Cells were pelleted in a microfuge and re-
suspended in nonreducing Laemmli buffer to a final volume normalized by
polyacrylamide gel (Bio-Rad, Hercules, CA). The samples were electro-
transferred to Immobilon polyvinylidene fluoride membranes (Millipore,
Billerica, MA) at 100 mV for 70 min. Western blot analysis was performed
using primary antibodies against the MscL C-terminus as previously de-
to the manufacturer’s instructions. X-ray-sensitive film (Blue Bio Film,
Denville Scientific, Metuchen, NJ) was exposed to the blotted membranes.
Quantification was done by measuring the density of the bands using Scion
Image software (NIH).
E. coli giant spheroplasts were generated and used in patch-clamp experi-
ments as described previously (23). Excised, inside-out patches were ex-
amined at room temperature under symmetrical conditions using a buffer
pH 6 (Sigma, St. Louis, MO). Recordings were performed at ?20 mV
(positive pipette). Data were acquired at a sampling rate of 20 kHz with a
software (Axon Instruments, Union City, CA). A piezoelectric pressure
transducer (World Precision Instruments, Sarasota, FL) was used to monitor
MscS was used as an internal standard for determining MscL sensitivity.
Measurements were performed using Clampfit9 from Pclamp9 (Axon In-
In vivo functional assays
Colonies were grown overnight at 37?C in citrate phosphate medium plus
1 mM ampicillin. The overnight culture was diluted 1:20 in this defined
medium, grown for l h, and then diluted to an OD600of 0.05 in the same
medium supplemented with 0.5 M NaCl. After one or two divisions, ex-
pression was induced for 30 min with 1 mM IPTG. The induced cultures
were diluted 1:20 into 1), citrate-phosphate medium containing 0.5 M NaCl
(mock shock) or 2), water (osmotic downshock). The in vivo modified
SCAM was performed in the presence of 1 mM MTSET or MTSES in the
downshock and mock shock solutions. Cells were then incubated at 37?C for
20 min, and then six consecutive 1:10 serial dilutions were made in medium
containing either no salt (for the osmotic downshock conditions) or 0.5 M
NaCl (for the mock-shock conditions). These diluted cultures were plated
and grown overnight, and the colony-forming units were counted and av-
eraged per experiment.
The S1 domain of MscL is critical for function
but more tolerant of substitutions than the TMD1
Previous studies (24,25) have demonstrated that deletions in
the N-terminal regionofMscL arepoorlytolerated. Channels
with a deletion of three amino acids (D2–4) are functional as
assayed by patch clamp, but deletion of 11 amino acids (D2–
12)led tononfunctionalchannels (24). Here,we extend these
in vivo assay. For this functional characterization, we have
matic representations of M. tuberculosis (left) and E. coli
(right) MscL structural models are shown. The most
N-terminal region of the channel (amino acids 2 to12) is
shown in red, and a single subunit in green for clarity. On
the left side of the panel, a model for a closed or nearly
closed structure of M. tuberculosis MscL derived from the
recently revised crystal structure (15) shows the homo-
pentameric nature of the channel with the N-terminal
region positioned at the cytoplasmic interface of the mem-
brane. Side (upper left) and cytoplasmic (lower left) views
are shown. The closed, closed-expanded, and open states
from the proposed Sukharev-Guy (SG) model for E. coli
MscL gating are shown in the three right-most panels (14);
side (upper row) and periplasmic (lower row) views are
shown. As shown in the middle of these panels (closed-
expanded), the model predicts the N-terminal region
(called S1) to play the role of a ‘‘second gate’’ that remains
closed after most of the expansion of the channel has
already occurred. The approximate membrane location is
indicated by the parallel gray lines in the side view.
Current models for MscL structure. Sche-
2284Iscla et al.
Biophysical Journal 95(5) 2283–2291
used the E. coli strain MJF455 (Dmscs/Dmscl), which has a
reduced viability after osmotic downshock and can be res-
cued by the expression in trans of wild-type MscL (18). The
ability of the deletion mutants to rescue this osmotically
sensitive strain compared with the wild-type channel shows
not only that D2–12 is nonfunctional but that even D2–4
has compromised function (Fig. 2 A). The D2–12 mutant
shows lower expression as determined by Western blot,
which could conceivably lead to its apparent nonfunctional
amount to only two to six functional channels per cell (23),
can save this osmotic-lysis phenotype and can even effect a
gain-of-function phenotype for some mutants (20). Given
these latter findings, and the fact that no one has yet observed
any channel activity by patch clamp for D2–12 (24,25), the
likely to be simply lower expression. In any case, one cannot
deny that the S1 domain is critical for function.
To determine whether a subdomain or single residue
within this region is of critical importance, residues 2–12 of
E. coli MscL were substituted with cysteines, and the phys-
iological propertiesofeachmutant determinedbyinvivoand
electrophysiological functional assays. A decrease in sta-
tionary phase OD600has previously been shown to be a
sensitive assay for channels that appear to gate inappropri-
ately in vivo (16). As shown in Fig. 3 A, of all S1 cysteine-
substituted channels, only cells expressing mutants R8C,
E9C, A11C, and M12C showed a small but statistically
significant decrease in their stationary phase OD600when
compared with wild type (lower dashed line). However, no
mutant caused a reduction greater than the 50% (upper
dashed line) that was a cutoff value used to designate a sig-
nificant functional deficit in a previous study of TMD1;
cells expressing eight different cysteine substitutions within
TMD1 exceeded this 50% reduction is growth (16). In ad-
dition, no obvious differences in the growth rates were ob-
served for any of the S1 mutants (data not shown). These
data suggest that none of the mutated channels had as high
a propensity for gating inappropriately in vivo as has been
previously observed for the pore-forming TMD1 region(16).
An in vivo functional assay similar to that shown in Fig.
2 A was performed on the S1 cysteine-substituted mutants to
assess their ability to rescue the osmotically fragile MJF455
strain. As shown in Fig. 3 B, all of the S1 mutants rescued the
vector (negative control, lower dashed line); however, most
mutants showed only a ‘‘partial’’ rescue of the lyses phe-
notype when compared with the expression in trans of wild-
type MscL (upper dashed line), suggesting that the mutated
MscL channels required more membrane tension than wild
type for gating. It is important to note that a growth pheno-
much of the decreased rescue observed for mutants R8C and
E9C (Fig. 3 B) could be because the cells are stressed from
expressing these mutated proteins (as indicated in Fig. 3 A).
In sum, although none of the cysteine mutations in the S1
domain leads to nonfunctional MscL channels in vivo, the
integrity of the region is required for entirely normal channel
function. Hereagain, thefindingthat noneofthechannelsare
nonfunctional contrasts similar cysteine-scanning studies of
the TMD1, where five mutants have been found to be non-
Because cysteine is a reactive residue, we can post-
translationally modify the cysteine-substituted amino acids
using sulfhydryl reagents. Previous studies have demon-
strated that when the MscL channel is gated in vivo, even
many cytoplasmicresiduesaresusceptible tomodificationby
the positively charged sulfhydryl reagent MTSET (2-(tri-
methylammonium)ethyl methanethiosulfonate) as evidenced
by a decreased viability; this has been coined as an in vivo
substituted cysteine accessibility method (in vivo SCAM)
(20,26). The viability of osmotically challenged cells (lead-
ing to gated channels) treated with MTSET, or the negatively
charged MTSES (2-sulfonatoethyl methanethiosulfonate), is
shown in Fig. 3 C, where the heights of the bars reflect the
difference in survival compared with the untreated cells. A
significant effect of the binding of MTS reagents on channel
function was observed only for mutants I4C, K5C, and E9C;
no dependence of charge was observed. Assuming the resi-
duesare accessible, these datawould be consistent with those
to a protein that is still expressed in the membrane but has a compromised
function. (A) The ability of the deletion mutants D2–4 and D2–12, when
expressed in trans, to rescue the MJF455 osmotic downshock-sensitive
strain in vivo was compared with that of cells expressing wild-type or empty
vector. Deletion mutant D2–4 partially rescued the osmotic-lyses phenotype,
whereasD2–12proved to be a nonfunctional channel.(B) Protein expression
of wild type and both deletion mutants was analyzed by Western blot in
whole cell (W), cytosol (C), and membrane fractions (M). Samples were
obtained from PB104 cells expressing each of the mutants. Most of the
protein was found in membrane fractions.
Deletion of the most N-terminal region of E. coli MscL leads
Structure of the N-Terminal of MscL2285
Biophysical Journal 95(5) 2283–2291
of the cysteine scan, which shows that no single residue ap-
pears critical. Only 27% of the S1 residues are sensitive to
these sulfhydryl reagents, which contrasts the finding that
40% (12 of 30) of the residues within and close to the TMD1
domain havebeen foundto beextremely sensitive toMTSET
(20,26). Hence, S1 appears to be much more resilient to
residue substitution or modification than are the adjacent
Finally, single-channel activities were characterized by
patch-clamp experiments in bacterial membranes by utilizing
giant spheroplasts. The activation pressure threshold of each
mutant was compared with that of MscS; the resulting ratios
are shown in Fig. 3 D. All the channels were functional, with
most of the mutants showing near-normal activation thresh-
olds when compared with wild-type MscL, confirming the
results of the in vivo functional experiments. Substitution
mutants E6C and F7C, followed by I3 and F10, showed the
most severe phenotypes, requiring the highest tensions to
gate. This pattern (residues 3, 6/7, and 10) is consistent with
ana-helix, which is atthe basis ofboth models.Overall, both
the in vivo and channel phenotypes observed here are subtle
when compared with substitutions in the both TMDs from
previous studies (16,19,21).
The disulfide bridging pattern and the effect
of oxidizing agents on channel function of the
cysteine mutants of S1 domain support the
Tb-MscL crystal structure
The ability of each of the substituted residues to interact with
their counterparts from neighboring subunits and form di-
mers was analyzed in vivo. These analyses were performed
subsequenttoosmoticdownshock and mockshock conditions
(in which MscL had been gated or not gated, respectively).
Samples derived from mock or osmotic downshocked bac-
terial cells expressing each of the S1 cysteine-substituted
channels were fractionated by SDS-PAGE and analyzed by
Western blot. Representative examples for each of the mu-
tants are shown in Fig. 4 A,in which thebandscorresponding
to the monomer and dimers are indicated by an arrow. The
bar graph in Fig. 4 B shows the average percentage of the
total protein that appears as a dimer. Note that there is no
significant disulfide bridging observed in this region unless
the cells are osmotically shocked. Given this result, it seems
unlikely that in vivo disulfide bridging, leading to locked-
mutants demonstrates that substitutions in the region are well tolerated.
(A) Little or no reduction in the stationary phase values was observed for
mutants when compared with wild-type MscL (lower dashed line). The
dashed line designates the 50% cutoff value used in previous studies to
designate a significant functional difference; none of the mutants studied
here achieved this value. (B) The ability of the S1 cysteine-substituted
mutants to rescue an osmotic-lysis phenotype is shown as a percentage of
survival. All of the mutated channels are functional when compared with
negative controls (vector only, lower dashed line); however, many of the
cysteine substitutions in the S1 region effected only a partial suppression of
the osmotic-lysis phenotype when compared with the wild type (upper
dashed line). (C) An in vivo SCAM study was performed for the S1 cysteine
mutants. The change in their ability to rescue an osmotic-lysis phenotype in
In vivo and patch-clamp characterization of S1 cysteine
the presence of MTSET (black bars) and MTSES (white bars) reagents is
shown in the graph as the weighted fold difference ((treated ? untreated)/
untreated). (D) Single-channel activities of each of the S1 mutants were
analyzed by patch clamp in giant spheroplasts. A Dmscl bacterial strain
(PB104) with an intact MscS was used. The pressure thresholds for the
activation of each of the MscL S1 mutants was compared with that of the
internal control MscS and expressed as a ratio.
2286 Iscla et al.
Biophysical Journal 95(5) 2283–2291
closed channels, is the reason many of the channels showed
only partial rescue of the osmotically sensitive strain, as
discussed above and shown in Fig. 3 B. Although the disul-
fide bridging observed on gating could be a result of struc-
tural movements occurring in the gating process, perhaps a
more likely explanation is that this is caused by a cellular
change in redox: the normally reductive cytoplasmic envi-
ronmentprobablybecomesmore oxidativeon channel gating
because of the cytoplasmic loss of glutathione and other re-
ducing agents such as thioredoxin (27). Results of identical
experiments performed with the adjacent S1-to-TMD1 linker
region (21) are also shown in Fig. 4 B for a better interpre-
tation of the data. Note that fewer dimers are observed in the
S1 domain than in this adjacent linker region.
of neighboring subunits should be inversely proportional to
the observed amount of disulfide bridging. In an attempt to
find a correlation between the disulfide bridging pattern and
the predicted distances by the two existing models, the in-
verse distances (1/A˚) were plotted in the same figure as line
graphs (open circles for SG Eco-MscL model and solid tri-
angles for Tb-MscL crystal structure). Residues predicted to
be in the closest proximity by the SG model and previously
shown to disulfide bridge (I3C, F7C, and F10C (13,14)) are
shaded inthefigure;these residuesdidnotshow thestrongest
interactions in the region, and no correlation was found be-
tween the disulfide bridging pattern observed and the pre-
dicted distances by the SG model. In contrast, there appeared
to be a clear correlation between the distances predicted by
the Tb-MscL crystal structure (15) and the amount of di-
In the original publication, the SG model was supported
not only by the ability to disulfide bridge residues I3, F7, and
F10 but also by the observation that the functional activities
of these channels were apparently sensitive to redox condi-
tions. Given the discrepancy between the disulfide bridging
pattern in Fig. 3 B and the predictions of the SG model, we
R13C, which has previously been demonstrated to be ex-
tremely sensitive to disulfide bridging and to achieve a
locked-closed conformation with this covalent linkage (16),
was used as a control. As expected for the low efficiency of
disulfide bridging observed for mutants I3C and F7C, their
activities did not show any evident changes between ambient
conditions andthe presence of anoxidant(H2O2) (Fig. 4) ora
reducing agent (DTT) (not shown). In contrast, F10C and
R13C, which showed a higher percentage of dimer forma-
tion, were influenced by redox. A decrease in the amplitude
of single-channel events was observed under oxidizing (and
even ambient) conditions for F10C, with full openings ob-
served more consistently in the presence of the reducing
agent DTT. However, this mutant was not locked closed
under these oxidizing conditions, and no changes in sensi-
tivity were observed among the different redox environments
used. As expected from a previous study (16), the most
drastic effects of oxidation on channel activity were observed
for R13C. Together, these data are not consistent with pre-
vious findings or the SG model (13,14). On the other hand,
the data are what one would expect if the S1 domain re-
mained along the membrane surface: residues closer to the
pore wouldbe more likely to be trapped by disulfide bridging
and effect a locked-closed channel phenotype.
ments show weak intersubunit interactions in
S1 cysteine mutants relative to the adjoining
ern blots for each of the cysteine-substituted
mutants are shown. Samples derived from bac-
terial cells diluted in a medium of the same
osmolarity (m for mock shock) or in water (o for
osmotic downshock) were fractionated by SDS-
PAGE, and MscL detected by Western blot. (B)
The bar graph (left y axis) shows the average
and standard error of the percentage total pro-
tein that is in the form of dimers for mock shock
(dark bars) or osmotically challenged (white
bars) samples derived from at least five exper-
iments similar to the examples shown in A. The
line graph reflects the inverse distances between
a-carbons of neighboring subunits for each
residue, as predicted by the model of M. tuber-
culosis (Tb-MscL, solid triangles) or by the SG
model for the closed E. coli MscL (Eco-MscL,
open circles). Note that smaller values represent
longer predicted distances. Results for the ad-
jacent region (S1-TMD1 linker region, residues
R13–D18) derived from similar experiments
In vivo disulfide-trapping experi-
using an identical protocol (21) are also shown to better determine which model best fits the data. Circled residues are those predicted by the SG model to
be in close proximity in the closed and closed-expanded conformations and were shown to yield strong disulfide bridging in membrane preparations (13).
Structure of the N-Terminal of MscL2287
Biophysical Journal 95(5) 2283–2291
Both the conservation of the region and functional studies
underline the importance of the S1 domain and in part led to
the speculation that this domain plays the role of a second
gate. This region of E. coli MscL is greatly conserved among
other bacterial species. In particular, phenylalanines 7 and 10
are conserved in 97% and 100%, respectively, in an align-
ment of 232 species (Supplementary Material, Data S1) and
are substituted only by leucines. The S1 domain motif of
XXYYFYYFXX, with X being hydrophobic and Y polar
amino acids, is conserved in 79%, and the outliers had only
subtle variations. Interestingly, the XXYYFYYFXX motif is
conserved even in species that have as many as 30 additional
amino acids at the N-terminal, further emphasizing its im-
portance in channel function. In addition, electrophysiolog-
ical studies found that even small deletions in this region (3,
8, or 11 amino acids) or substitution of 9 amino acids of the
S1 domain with a random sequence lead to channels with
either a decreased sensitivity or total loss of activity (24,25).
Here we further characterized in vivo two deletion mutants
and find that deletion of 11 residues is sufficient to lead to a
nonfunctional, but membrane-associated, protein, and the
deletion of as few as three residues measurably compromises
function (Fig. 2). Unfortunately, this critically important re-
gion of the protein was not resolved in the first Tb-MscL
crystallographic structure (5), and one could only speculate
channel, it was noted that a large amount of energy was re-
quired to effect MscL gating. Because this energy translated
into a change in area (DA), Sukharev et al. (28) interpreted
these data as supporting a model in which the channel ex-
pands within the plane of the membrane before ion permea-
tion; the critical yet unresolved N-terminal region of the
protein seemed a logical location of a second gate that would
allow for this expansion (Fig. 1). The authors performed
targeted disulfide-trapping experiments, which seemed to
confirm their supposition (13,14).
Although appealing, the SG model did not easily account
for some experimental data, thus leading to the proposal of
et al. (11) for a review). Mutations in TMD1 that were found
to increase channel sensitivity also decreased the open dwell
principal gate but that it is the transient exposure of hydro-
phobic regions in this domain to the pore lumen that is the
primary energy barrier to achieving an open structure; mu-
tation to more hydrophilic residues decreases this energy
barrier, leading not only to more sensitive channels but to
activities that more easily transition between closed and open
states (1,16,29). If S1 truly formed a second gate, one might
expect that mutations here would also lead to channels that
misfunction. But random mutagenesis combinedwithin vivo
assays showed that there is a great tolerance for mutations in
this region (19,30,31). Here we have used a more systematic
and sensitive approach and find that substitutions at many
sites within this domain do indeed lead to phenotypes, albeit
subtle, when compared with those of TMD1 (16), thus sup-
porting the functional importance of the region. Although the
with the SG model, the locations of some of the phenotype-
effecting mutations (e.g., R8C and E9C) do not give strong
support for it either. Structurally the S1 domain is predicted
by the SG model to be a helical bundle; however, a recent
reevaluation of the Tb-MscL crystal structure allowed for the
ine mutants under different oxidizing condi-
tions. Representative traces of single-channel
activity for mutants I3C, F7C, F10C, and R13C
shown under different redox conditions. Re-
cordings were made at ?20 mV, and by con-
vention openings are shown as downward. No
differences were observed in channel activity
for I3C and F7C mutants measured before
(ambient, left) and after treatment with the
oxidant H2O2 (right). Unlike I3C and F7C,
F10C and R13C showed variable activity under
ambient conditions and were influenced by
DTT. Therefore, these membrane patches were
treated with DTT for 5 to 35 min (left) before
oxidative treatment (right). Note that a decrease
in the amplitude of F10C was observed in
presence of H2O2, whereas R13C activity was
abolished under identical oxidizing conditions.
All H2O2-treated traces reflect activities 5 to 15
min after the addition of oxidant.
Single-channel activity of cyste-
2288 Iscla et al.
Biophysical Journal 95(5) 2283–2291
resolution of the S1 domain (15), and the new structure
showed the domain along the cytoplasmic membrane inter-
face. Here again, this is not strong evidence against the
SG model because, given that there are most likely several
closed states of the channel, the SG closed structure could be
achieved only under some conditions (e.g., on initial tension
in the membrane). Although none of these observations by
itself strongly disputes the SG model, the explanations re-
quired to accommodate the findings sometimes seem strained;
we therefore believed that it was appropriate to directly re-
evaluate the data underlying the SG model.
The SG model predicted specific protein-protein interac-
tions within the proposedS1bundle, which were tested in the
original publication using targeted disulfide trapping of I3C,
F7C, and F10C in isolated membranes (13,14). We have
repeated these experiments but have assessed the state of the
PAGE loading buffer), thus avoiding any manipulations
before Western blot analysis. In addition, we have compared
residues within the entire region and even an adjacent region
that we characterized using an identical approach (Fig. 4)
(21). We found that F7C did not form measurable disulfide
bridges, and although residues I3, and F10 do, they were not
as reactive as neighboring residues; instead, it appeared that
the hydrophilic face of the amphipathic helix was the most
reactive (K5 . I3, E6 . F7, and E9 . F10). In addition, the
pattern of disulfide bridging across the region is not consis-
tentwiththat predicted bytheSGmodel butreflectswhatone
might expect for the current Tb-MscL structure: residues
more distal to the pore had, in general, a smaller probability
of disulfide bridging. Another strong argument supporting
the SG model in the original article was the finding that F7C
and F10C activity was not observed in patch clamp except
when treated with reducing reagents (13). In contrast to this
previous report, here we were able to demonstrate channel
activities in both cases independent of reducing agents; al-
though F10C activity is clearly modified by redox, H2O2
never completely locked the channel closed, and we saw no
effect of redox on F7C. Similarly, the original report for the
SG model stated that the I3C channel partially closed when
treated with an oxidizing reagent; again, our data do not
agree. The most likely reason for this discrepancy is the
difference in the experimental conditions used. The original
report treated the patch with I2for 30 min, whereas here we
use the less-aggressive oxidant, H2O2, for 5 to 15 min, which
appears to besufficient(see thepositive controlR13C). From
the data presented in Fig. 5, it again becomes apparent that
disulfide bridging that affects channel function is greatest
when near TMD1 (R13C) and decreases distally. Again,
these data support the model in which the S1 domain lies
along the cytoplasmic membrane.
Collectively, our data support the newly reevaluated Tb-
MscL crystal structure, placing S1 along the cytoplasmic
surface ofthe membrane (15). But what could be the function
of such a structure? In determining the function of the S1
domain, it may be important to note that all current models
for the gating of MscL predict a significant tilting of the
TMD1 membrane on opening (13,14,32). We propose that
the S1 domain helps to define this tilt by maintaining its in-
teraction with the membrane by serving as an anchor (Fig. 6).
Interestingly, the two most conserved residues within the S1
domain, F7 and F10, represented as diamonds in Fig. 6, are
aromatic and hydrophobic; thus, they have a high affinity for
the lipid environment. This could also explain why in a few
instances these residues are not conserved; the substitution is
always the hydrophobic residue leucine. The functional role
ofG14asahinge between TMD1 andS1domainswouldstill
be critical for channel function, as previously shown (13).
far from several studies, including the apparent inconsistency
between deletion and missense mutations. It may be of in-
terest to note that a similar structure is found in the bacterial
inward-rectifying K1channel KirBac, in which a cytoplas-
mic a-helix running parallel to the membrane (slide-helix)
(33,34) was found to interact directly with the phospholipid
headgroups to regulate channel gating (35). In another study,
the aromatic nature of a residue at the cytoplasmic end of
the putative pore-forming TMD6 domain of the MS yeast
TRPY1 channel has been shown to be important in gating
and has been referred to as a ‘‘gate anchor’’ (36); the region
could potentially form an amphipathic a-helix, although the
structure ofthischannelhasnotyetbeen determined.Finally,
a crystallographic structure of MscS, another bacterial MS
channel from an independent channel family, also appears to
contain an a-helix along the cytoplasmic/membrane surface
just adjacent to the pore (4,15). Similar to MscL, current
schemeshowsthe S1andTMD1domains ofa singlesubunitof MscLbefore
(left) and after (right) applying tension to the membrane. The upper graph
shows a wild-type MscL where the S1 domain is intact. The two most
conserved residues in this region, F7 and F10, are shown as pentagons along
this structure, stabilizing the interactions with the membrane. The region
serves as an anchor for TMD1 to the cytoplasmic side of the membrane,
facilitating and guiding the tilting of TMD1 on membrane-tension-effected
bilayer thinning and channel opening. The lower graph shows an S1 deletion
mutant of MscL. The lack of this anchor region impairs the proper tilting of
the TMD1 domains needed for channel gating, so the channel remains
closed. Tilting angles shown for the TMD1 domains in the closed and open
states were derivedfrom the crystal structure for Tb-MscL and the open state
of E. coli MscL from the SG model, respectively.
Model for the role of the S1 domain in MscL gating. The
Structure of the N-Terminal of MscL2289
Biophysical Journal 95(5) 2283–2291
models of MscS gating predict a tilting of the pore domain
(37), and a glycine is also presentbetweenthese two domains
(Gly-113), probably serving as a hinge (4,15). Thus, it is
tempting to speculate that this analogous domain within
MscS, and perhaps other channels, may serve a similar
function as the S1 domain in MscL.
To view all of the supplemental files associated with this
article, visit www.biophysj.org.
The work was supported by Grant I-1420 of the Welch Foundation, Grant
of the American Heart Association—Texas Affiliate, and Grant GM61028
from the National Institutes of Health.
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Structure of the N-Terminal of MscL2291
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