www.sciencemag.org/cgi/content/full/321/5893/1210/DC1

Supporting Online Material for

A Structural Mechanism for MscS Gating in Lipid Bilayers Valeria Vásquez, Marcos Sotomayor, Julio Cordero-Morales, Klaus Schulten, Eduardo

Perozo*

*To whom correspondence should be addressed. E-mail: eperozo@uchicago.edu

Published 29 August 2008, Science 321, 1210 (2008) DOI: 10.1126/science.1159674

This PDF file includes:

Materials and Methods Figs. S1 to S6 Table S1 References

1

Supplementary Materials

A Structural Mechanism for MscS Gating in Lipid

Bilayers

Valeria Vásquez, Marcos Sotomayor, Julio Cordero-Morales, Klaus Schulten, and

Eduardo Perozo.

Materials and Methods

Spheroplast Patch-Clamp. Channel activity was recorded by patch clamping giant

spheroplasts following well-established protocols (1, 2). Patch Clamp measurements

were done in the inside-out configuration, under symmetrical conditions (200 mM KCl,

90 mM MgCl2, 10 mM CaCl2, and 10mM HEPES, pH 7.5 at room temperature).

Spheroplasts integrity was maintained by using 500 mM sucrose in the pipette and bath

solutions.

Patch pipettes (SIGMA, catalog number P1174) were fire polished to resistances

between 2 and 2.5 MΩ. Negative pressure on the patch was obtained by applying suction

through a pressure-clamp system (HSPC-1, ALA Scientific Instruments). Liposome-

reconstituted channels were activated without the application of negative pressure, by

perfusing 3 μM of 18:1 1-Oleoyl-2-Hydroxy-sn-Glycero-3-Phosphocholine (LPC from

Avanti Polar Lipids) into the bath solution. Macroscopic currents were recorded with a

DAGAN 3900 patch-clamp amplifier, and currents were sampled at 10 KHz with an

analog filter set to 2 KHz. Analyses were done using pCLAMP9 (Axon Instruments).

Mutagenesis, expression, spin labeling, and reconstitution of MscS. Cysteine mutants

(residues 2-128) were generated for a previous study (3). Individual mutants covered the

2

N-terminus, and the three transmembrane helices of MscS. Mutant channels were

expressed, purified, labeled and reconstituted as previously described (4). The samples

were reconstituted at a 750:1 lipid/channel (DOPC:POPG, 6:1) molar ratio by dilution in

PBS in the presence of Bio-beads.

EPR spectroscopy and data analysis. X-band CW EPR spectra were obtained at room

temperature, as previously described (5). Spectra were obtained in a Bruker EMX

spectrometer fitted with a dielectric resonator under the following conditions: 2 mW

incident power, 100 kHz modulation frequency and 1 G modulation. The accessibility

parameters were quantified as reported (3, 5, 6).

Three-dimensional modeling of MscS TM segments in the open state. More than 30

starting conformations and refinement conditions were tested to generate a model for the

open state of MscS. In modeling the MscS open structure, a symmetric EPR-based model

of MscS in its closed state (3) was used as the starting conformation (residues 1 to 178).

A rigid-body rotation was applied to helices TM1, TM2, and TM3 (50o, 36o, and 130o,

respectively) according to the resultant vector angular differences between closed and

open states established by EPR spectroscopy (Fig. 3C and fig. S4). Helices were rotated

around their longest principal axis and a computational refinement of this initial

conformation was carried out as outlined in refs. 3, 7. Briefly, two virtual EPR particles

were harmonically attached to each C-α atom of the protein. The first particle, labeled

EPR, represented a spin-label probe and was attached through a bond with a resting

length of 6 Å. The second particle, labeled PROT, was attached on top of the C-α atom

of the corresponding residue. The complete system, including protein atoms and EPR

particles, was embedded in an environment of pseudo-atoms representing particles for the

3

contrast agents O2 and NiEdda. The distribution of these pseudo-atoms mimicked the

membrane location; an additional cylinder of NiEdda particles crossing the bilayer

represented a hydrated pore. The cylinder was enforced using the MMFP module of

CHARMM. The pseudo-spin probes were assigned attractive or repulsive interactions to

O2, NiEdda, and PROT particles based on experimentally determined EPR accessibilities

for each residue. Buried pseudo-spin probes were allowed to overlap with PROT

particles. The system was simulated using the CHARMM program version c32a2 (8)

with the extended atom PARAM19 force field modified to reduce the effect of charged

residues in vacuum. RMSD constraints maintaining the initial secondary structure were

applied to backbone atoms of residues 10-14, 20-26, 29-57, 68-91, 96-113, 114-128, and

129-178. In addition, a cylindrical harmonic potential was applied to all C-α atoms in

order to promote channel opening. Refinement was carried out through multiple cycles of

minimization and equilibration. The coordinates of the resulting model (C-α atoms) are

available at http://www.ks.uiuc.edu/Research/MscSchannel.

Molecular Dynamics Simulations. To test the stability and conduction properties of the

open MscS EPR-based model we conducted additional all-atom MD simulations in a

realistic fully hydrated membrane environment (see Table I). The simulated system

contained the refined open model of MscS (residues 1 to 178), a POPC lipid bilayer, 200

mM of KCl, 90 mM of MgCl2, and 10 mM of CaCl2 (166,395 atoms). The simulations

were carried out using NAMD2.6 (9), the CHARMM27 force-field (8) with the CMAP

correction (10), and the TIP3 model for water. Other simulations details are as described

in ref. 3.

4

Concentration dependence of LPC-induced conformational changes. To establish the

concentration-dependence of LPC-induced conformational changes, we focused on three

positions along the length of TM1 (N30C, L42C, and N50C), a region that allows for a

thorough spectroscopic coverage of the MscS TM domain (3). In contrast to the dramatic

structural changes observed in MscL (11), addition of LPC generated reproducible, but

subtler changes in the overall line shape of TM1 spin-labeled residues (fig. S1). Dose-

response curves (fig. S1, right) were obtained from changes in the mobility parameter,

where increasing LPC molar fraction in the bilayer generated a saturating increase in

probe mobility for positions N30C-SL and N50C-SL, with a mid-point around 8-9 %.

Although there is a significant loosening of the MscS structure, this effect is not due to

non-specific increases in global dynamics, as demonstrated by the relative insensitivity of

position L42C-SL to LPC incorporation (fig. S1, left).

5

Fig. S1. Upper left panel, CW-EPR spectra for TM1 N30C-SL, L42C-SL, and N50C-SL obtained at

increasing concentrations of LPC. Upper right panel, relative changes in spin-label mobility for N30C-SL

and N50C-SL as a function of LPC molar percent. Bottom panels, incorporation of 3 µM LPC from the

pipette side elicits MscS activation, even in the absence of applied negative pressure at +10 mV (left) and

-10 mV (right), respectively. The dotted line represents zero current level.

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Extent of environmental parameter changes of MscS upon addition of LPC

Fig. S2. Extent of environmental parameter changes of MscS upon addition of LPC. Δ represents open

minus closed state parameters. Top panel, changes in mobility (ΔΔHo-1). The red broken curve indicates

how major changes in mobility are located at the ends of TM1 (residues V29C-SL to L35C-SL, and S49C-

SL to I57C-SL). Middle panel, differences in O2 accessibility (ΔΠO2). Bottom panel, changes in NiEdda

accessibility (ΔΠNiEdda). Gray areas represent the TM segment assignment derived from the MscS crystal

structure (12, 13).

7

Residue environmental parameter profiles mapped onto molecular surfaces

To better appreciate the EPR results, we have also mapped the raw data set in the

experimentally determined three-dimensional models (see figure below). Mapping the

environmental parameters, onto the closed conformation model and the crystal structure

corroborates that several rearrangements take place in the LPC-induced open

conformation.

Fig. S3. Residue environmental parameter profiles mapped onto molecular surfaces of (A) the closed state

EPR-based model and (B) a non-conductive conformation (crystal structure). Left panel, ribbon

representation of MscS (two subunits are shown for clarity). Individual TM segments are color-coded as

follows: N-terminus, green, TM1, yellow; TM2, blue; and TM3, red. From left to right panels: Mobility

(ΔΗo-1), Oxygen accessibility (ΠO2), and NiEdda accessibility (ΠNiEdda).

8

Analysis of the Conformational Rearrangements during Activation Gating

We have used a quantitative approach to estimate the secondary structure from

our one-dimensional data set of reconstituted MscS upon incorporation of LPC. This

secondary assignment was used to build the EPR-based three-dimensional model of

MscS in an open conformation. This approach (3, 5, 14), is based on the estimation of the

angular frequency peaks (in Fourier space) present in a linear series of properties along a

given sequence of residues (15). This approach allows for the determination and

assignment of secondary structural elements, given the periodic behavior of a segment

based on EPR-derived environmental properties (14).

In the closed state, both TM1 and TM2 segments (but not TM3) displayed a

prominent peak within the α-helical region (3). We find that the overall periodicity

parameters of the α-helical structures remain essentially unchanged, a clear indication

that there is no general unwinding of the TM domain segments upon opening. It also

suggests that the main movements could occur as rigid bodies, with a few changes in

secondary structure. The TM3 segment, on the other hand, displays rather profound

changes. In the closed conformation, spin-labels along TM3 appear restricted by multiple

protein contacts; as such, no clear periodicity is revealed (fig. S4). However, upon

opening, when the pore diameter increases, the spin label displays a noticeable increase

in motional freedom and NiEdda accessibility. These changes lead to a well-defined peak

in the angular power spectrum at ~98o, and represent the strongest evidence that

asymmetric incorporation of LPC stabilized the open conformation of MscS.

9

Fig. S4. Calculation of the Fourier transform power spectrum of the accessibility profiles for the TM3 in

the closed and open conformations. The angular frequency range from 80o-120o corresponds to an α-helical

structure. Power spectrum of the TM3 segment ΠNiEdda profile. The black arrow shows the periodicity

gain of the pore segment upon opening.

To further examine the magnitude of the conformational changes upon opening,

we have superimposed the individual environmental moments for the spin-labeled

residues from the closed and open state on a helical wheel representation. In general, the

vector analysis in the context of a helical wheel projection indicates that the conformation

changes upon opening could be explained by rotation and tilting of the N-terminus and

TM helices.

10

Fig. S5. Frequency and vector analysis of environmental data point in the open conformation.

Environmental parameters have been superimposed in a polar coordinate representation. (A-D) Resultant

moments, for the closed (black arrow) and open (red arrow) conformations, were calculated for the

accessibilities of each TM segment and superimposed on a helical wheel of the N-terminus, TM1, and TM2

segments.

11

All-atom MD simulations of EPR based MscS model in the open

conformation

More than thirty models were generated using the pseudoatom-driven solvent

accessibility refinement approach (PaDSAR). Although several promising candidates

were obtained we chose the model that better satisfied our accessibility and mobility data.

The stability of the resulting model was probed through all-atom MD simulations in an

explicit, fully hydrated membrane environment (fig. S6).

Fig. S6. All-atom MD simulations of the EPR based model of MscS in its open conformation. (A)

Snapshots of the system after 4 ns of constrained dynamics (top left), and at the end of 21 ns of free

dynamics at 0 V (top right), +1 V (bottom left), and -1 V (bottom right). (B) RMSDs for the MscS EPR

model at different biasing potentials. Middle, ion-crossing events through the MscS EPR-driven open

12

model at +1 V. A conductance of 1.6 nS is estimated (see Table I). Bottom, ion-crossing events through the

MscS model at -1 V. A conductance of 0.8 nS is estimated (see Table I).

Table I

Label tsim (ns) Type Ensemble Voltage Start + charge +z +charge –z Cl- +z Cl- -z

Sim1 7.00 EQ NpT a 0 - - - - -

Sim2 16.24 EQ NpT 0 Sim1 - - - -

Sim3 18.08 V NpT +1V Sim1 0 66 140 2

Sim4 18.24 V NpT -1V Sim1 17 0 0 77

a This simulation consisted of multiple steps with the protein fixed and lately restrained (4 ns of dynamics),

followed by 3 ns of free dynamics.

Summary of simulations. All simulations were performed using 200 mM KCl, 90 mM

MgCl2, and 10 mM CaCl2. EQ denotes equilibrium simulations; V denotes simulations

performed using a biasing voltage. Initial coordinates and velocities were obtained from

the last frame of the simulations mentioned in the Start column.

13

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