Capturing the Membrane-Triggered Conformational Transition of an Î±

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Capturing the Membrane-Triggered Conformational Transition of an #-Helical Pore Forming Toxin V. V. Hemanth Giri Rao, Rajat Desikan, K Ganapathy Ayappa, and Shachi Gosavi J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b09400 • Publication Date (Web): 31 Oct 2016 Downloaded from http://pubs.acs.org on November 4, 2016

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The Journal of Physical Chemistry

Capturing the Membrane-Triggered Conformational Transition of an α-Helical Pore Forming Toxin V. V. Hemanth Giri Rao,†,¶ Rajat Desikan,‡,¶ K. Ganapathy Ayappa,∗,‡,§ and Shachi Gosavi∗,† Simons Centre for the Study of Living Machines, National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bengaluru, India, 560065, and Department of Chemical Engineering, Indian Institute of Science, Bengaluru, India, 560012 E-mail: [email protected]; [email protected] Phone: +91 (80) 2293 2769; +91 (80) 2366 6105. Fax: +91 (80) 2360 8121; +91 (80) 2363 6662

∗

To whom correspondence should be addressed Simons Centre for the Study of Living Machines, National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bengaluru, India, 560065 ‡ Department of Chemical Engineering, Indian Institute of Science, Bengaluru, India, 560012 ¶ These authors contributed equally to this article. § Centre for Biosystems Science and Engineering, Indian Institute of Science, Bengaluru, India, 560012 †

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Abstract E. coli Cytolysin A (ClyA) is an α-helical pore forming toxin (PFT) which lyses target cells by forming membrane permeabilizing pores. The rate determining step of this process is the conversion of the soluble ClyA monomer into a membrane inserted protomer. We elucidate the mechanism of this conformational transition using molecular dynamics simulations of coarse-grained models of ClyA and a membrane. We find that a membrane is necessary for the conformational conversion because membraneprotein interactions counteract the loss of the many intra-protein hydrophobic interactions that stabilize the membrane-inserting segments in the ClyA monomer. Of the two membrane-inserting segments, the flexible and highly hydrophobic β-tongue inserts first while the insertion of helix αA1 is membrane assisted. We conclude that the β-tongue is designed to behave as a quick response membrane sensor, while helix αA1 improves target selectivity for cholesterol containing cell membranes by acting as a fidelity check.

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Introduction Pore forming toxins (PFTs) are virulent proteins secreted by many pathogenic bacteria that afflict several organisms including humans. PFTs form non-selective transmembrane pores in the plasma membranes of target cells and cause diverse effects including disruption of intra-cellular ion concentrations and osmotic gradients, and cell death 1 . Structural evidence suggests that the PFT pores are oligomeric with membrane spanning subunits. These subunits have a different conformation from that of the originally secreted water soluble monomers 1 . Unlike PFTs, most transmembrane proteins do not have structurally or functionally well defined water soluble conformations. Therefore, PFTs offer interesting examples of membrane-triggered protein conformational transitions with lethal consequences for target cells. PFTs are primarily classified as α and β-PFTs, depending on whether their membraneinserted segments are predominantly α-helices or β-sheets 1 . Cytolysin A (ClyA) is an α-PFT secreted by pathogenic E. coli, S. enterica, S. flexneri, and S. aureaus strains 1–4 . Pore formation by ClyA does not require any post-translational modifications or a protein receptor on the target cell membrane, but is specific to membranes containing cholesterol 1,5 . E. coli ClyA (34 kDa, 303 residues) is one of the few α-PFTs whose water-soluble form 2 (monomer, Fig. 1A) and transmembrane pore 3 have both been structurally characterized. Upon membrane recognition and binding, the soluble monomer transforms into a membrane inserted conformation (protomer), which subsequently homo-oligomerizes to form dodecameric transmembrane pores 3,4,6 . The structure of the protomer is taken to be that of the transmembrane pore-subunit 3 and is shown in Fig. 1B. A recent single molecule Förster resonance energy transfer (smFRET) experiment suggests that the conformation of the transmembrane pore sub-unit and the protomer are similar 6 . The protomer has two membrane-inserted segments, residues 1 to 35 (helix αA1) and residues 177 to 203. We refer to the latter as the β-tongue 3 , due to its predominantly β-hairpin conformation (residues 180 to 195) in the monomer, even though it forms a helix-turn-helix motif in the protomer. Both membrane-inserted segments 2 ACS Paragon Plus Environment


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relinquish their intra-protein interactions in the monomer in order to insert into the membrane. The structures of the two conformations (Fig. 1) also suggest that the transition from the monomer to the protomer involves secondary structural (β-strand to α-helix and loop to α-helix) transformations, disruption and reorganization of inter-helix interfaces, and rearrangements of up to 140 Å 3 . This makes it the largest conformational transition observed in monomeric proteins of length ∼300 residues 3 . Pore formation by ClyA is an irreversible process occurring on a timescale of hundreds of seconds in which the conformational transition is rate-limiting at monomer concentrations higher than ∼1 µM 6,7 . However, the molecular details of this conformational transition are poorly understood due to limitations of structural resolution in experiments 6 , and inaccessibility of long time-scales in all-atom, explicit solvent molecular dynamics (MD) simulations. In this study, we use MD simulations of a coarse-grained structure based model (SBM) of ClyA along with a coarse-grained membrane model to provide molecular details of how the soluble monomer transitions into a membrane inserted protomer. SBMs capture the funnel-shaped energy landscape of proteins by encoding the native structure of the protein into a coarse-grained potential energy function 8,9 . These models, when extended to encode two structures in the potential energy function, are termed dual SBMs. Dual SBMs are based on the assumption that the structures of the two conformations determine the mechanism of conformational transition. MD simulations of dual SBMs have been successful at capturing the long time and large length scale conformational transitions of both soluble 10–13 and transmembrane 14 proteins in computationally tractable timescales. In this study, we simulated a dual SBM of ClyA coarse-grained to a Cα representation which encodes the structures of both the monomer and the protomer, and found that this model does not undergo the conformational transition. In order to capture this transition, it was necessary to construct a coarse-grained membrane model that encodes membrane-protein interactions and is compatible with Cα dual SBMs. Even though biological membranes are complex multicomponent systems 15 , coarse-grained lipid bilayers which capture essen-


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tial membrane-protein interactions have been successfully used to represent the membrane environment surrounding transmembrane proteins 15–21 . In our model, we identify the minimal membrane-protein interactions required for the conformational transition of ClyA. This approach allowed us to sufficiently sample the conformational landscape of ClyA on computationally reasonable timescales. Additionally, the effects of desolvation, electrostatics, protein side-chains, membrane fluidity and curvature can also be included in scenarios where such interactions may be important. We find that the interactions between the membrane and the two segments which are membrane-inserted in the ClyA protomer facilitate the conformational transition from the monomer to the membrane inserted protomer. The observed mechanism of this membranetriggered conformational transition is explained by the monomer specific structural features of these segments. Interestingly, this structural rationalization also provides an explanation for the rate-limiting nature of this transition as observed in experiments 6,7 . In addition, our simulations indicate that one membrane-inserted segment of ClyA, the β-tongue, is designed to behave as a quick response membrane sensor while the other membrane-inserted segment, helix αA1, acts as a potential fidelity check. By using a simple membrane model which only accounts for the structurally evident membrane-protein interactions of the protomer, we not only captured the monomer to protomer transition, but also found that membrane-like features are required but only to the extent that they sufficiently stabilize the protomer.



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Figure 1: A) Structure of the monomer (PDB 1QOY). We define the head domain and the tail domain according to a previous study 3 . The segments between these two domains are termed here as the middle domain. Cα atoms of residues at the boundary of these domains are marked by black spheres. Helices αA1 (cyan, residues 1-35), αA2 (purple), αB (lime green), αC (olive green), αF (pink), αG (red), and the β-tongue (green, residues 177-203), are colored and marked. The rest of the protein is colored in grey. Notably, helix αA1 is an integral part of the tail domain, while the β-tongue is predominantly a β-hairpin (residues 180-195) in the head domain. The membrane-triggered conformational transition involves the swinging out of helix αA1 and the β-tongue, and these are marked by cyan and green arrows respectively. B) The structure of the protomer (PDB 2WCD, chain A) shows the two membrane-inserted segments, i.e. helix αA1 and β-tongue, as well as the extra-cellular segments according to the color scheme in A.


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Materials and Methods Cα SBM Here, we used a Cα SBM in which the protein is represented only by its Cα atoms. The potential energy function of a Cα SBM is given by the equations below 8,22 , and is exclusively composed of interactions between Cα atoms. n=1,3

Ubonded =

X

2

Kr (r − r0 ) +

bonds

Unonbonded =

X

2

Kθ (θ − θ0 ) +

angles

X

[(1 + (

contacts

X

(n)

Kφ (1 − cos(n(φ − φ0 )))

(1)

dihedrals

σN C 12 ) )(1 + G(rij , roij )) − 1] rij

X

+

non−contacts

(

σN C 12 ) rij

where G(rij , roij ) = −exp[−(rij − roij )2 /(2σ 2 )]

(2) (3)

The first and second terms in Ubonded represent bond vibration and angle bending motions through harmonic potentials, and the third term represents dihedral angle rotation using a combination of two cosine functions. The equilibrium bond lengths (r0 ), bond angles (θ0 ), and dihedrals (φ0 ), are calculated from the Cα atom coordinates obtained from the native (1)

structure. The force constants are Kr =10000 kJ/mol-nm2 , Kθ =20 kJ/mol-rad2 , Kφ = and (3)

Kφ =0.5, where is the energy scale and is equal to one reduced unit of energy 23 . Since we perform simulations using GROMACS which uses kJ/mol as the basic unit of energy, we set the value of to 1 kJ/mol. The first term in Unonbonded is a Gaussian potential that represents interactions between Cα atoms of residue pairs that form native contacts. For every pair of Cα atoms i and j which form a native contact, σN C (= 0.36 nm) is the excluded volume interaction distance, rij is the distance between the two atoms and σ (= 0.05 nm) is the width of the Gaussian contact well that has a minimum at roij (contact distance observed in the native structure) and a depth of −. Unlike an LJ potential, the Gaussian potential decouples the excluded volume interaction from the native contact interaction, and both can now be varied independently



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of each other. The second term in Unonbonded represents the excluded volume interaction between non-contact pairs i.e. Cα atoms of residue pairs that are neither in covalent nor in non-covalent interactions in the native structure. Together, Ubonded and Unonbonded encode interactions in such a way that the protein has the lowest potential energy when its Cα atoms have coordinates corresponding to the native structure.

Cα dual SBM We constructed a dual SBM by extending the Cα SBM potential energy function to encode two conformations of a protein instead of one native structure. Since the equilibrium bond lengths between two Cα atoms is the same (∼0.38 nm) irrespective of protein conformation, bond vibrations are represented with single well potentials as above. Dual potential functions for angle bending, dihedral angle rotation and native contacts are described below.

Dual angle potential A dual harmonic potential was constructed as published previously 12 , with a spring constant Kθ and two minima, θ1 and θ2 , corresponding to the two native angles in the two conformations of the protein (Fig. 2A, red curve).

Udual−angle = Kθ (θ − θ1 )2 (θ − θ2 )2

(4)

Dual dihedral potential The dual dihedral potential published previously 12 defined two barriers between the two native dihedrals observed in the two conformations of the protein, such that a barrier was encountered when going from one native dihedral to the other in both increasing and decreasing values of the dihedral angle. We modified this potential further so that these two barriers were similar to each other. The essential functional form remained the same, but we used multiple, differently scaled versions of the same functional form arranged in a piece-wise 7 ACS Paragon Plus Environment

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continuous manner, as shown below (illustrated in Fig. 2B, red curve).

1

(λ2 − λ1 )J(φ) ;

φC ≤ φ < φ1

(5)

;

φ1 ≤ φ ≤ φ2

(6)

(λ1 − λ2 )J(φ) ; 1+ φ1 − φ2 2 φ1 + φ2 )) − cos( )] J(φ) = [cos(φ − ( 2 2

φ2 < φ ≤ φD

(7)

Udual−dih = λ1 J(φ) +

1 + e−l(φ−φA )

= λ3 J(φ) 1

= λ2 J(φ) +

l = 10 ;

λ1 = 1 ;

e−l(φ−φB )

λ2 = 2 ;

(8)

3(φ −φ ) 1 1 − cos( 12 2 ) 1 1 + ] λ3 = [ 2 2 2 1 − cos( φ1 −φ ) 2 (1 − cos( φ1 −φ ))2 2 2

(9)

For a given dihedral angle rotation, φ1 and φ2 refer to the two native dihedral angles observed in the two conformations, φC =

φ1 +φ2 −2π , 2

φD = φC + 2π. φA and φB are the

location of the switching functions and are defined as φA =

3φ1 +φ2 −2π , 4

φB =

φ1 +3φ2 +2π . 4

λ1 ,

λ2 and λ3 refer to the force constants that dictate the steepness of the potential and the barrier height at

φ1 +φ2 . 2

Dual contact potential The Gaussian contact potential (Fig. 2C) was used to define both single and dual contact potentials as published previously 22 .

Udual−Gaussian = [(1 + (

σN C 12 ) )(1 + G(rij , r1ij ))(1 + G(rij , r2ij )) − 1] rij

(10)

where G(rij , r1ij ) = −exp[−(rij − r1ij )2 /(2σ12 )]

(11)

where G(rij , r2ij ) = −exp[−(rij − r2ij )2 /(2σ22 )]

(12)

For every contact, σN C , and rij are as defined in Eq 2. r1ij and r2ij are the native contact distances observed in the two conformations. The two Gaussian wells have a width of 0.05 nm (= σ1 = σ2 ) and a depth of −. 8 ACS Paragon Plus Environment


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Figure 2: Dual potential functions for angles, dihedrals and contacts. A) The dual angle, B) dual dihedral and C) dual contact potentials are shown in red, while the two corresponding single potentials are shown in black (solid and dotted). D) For a single contact potential (black), the formation of the native contact with increasing r is shown in grey. Only if the corresponding pair of Cα atoms are at a distance less than or equal to the contact minima, their contact contributes +1 to the number of native contacts formed, else the contribution from their contact decreases to 0 continuously with increasing distance (grey).


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Cα dual SBM of ClyA The structures of ClyA in the monomer and protomer conformations were obtained from PDBs 1QOY 2 and 2WCD (chain A) 3 respectively. Residues 299 to 303 (amino acid sequence EVPEV) were missing in 1QOY, while residues 1 to 7 (amino acid sequence MTEIVAD) and 293 to 303 (amino acid sequence GKKTLFEVPEV) were missing in 2WCD (chain A). These residues were modeled as loops using Modeller 9.9 24 while keeping the remaining atoms fixed. In addition, mutations A2T and V187A were also made to 1QOY so that both conformations now have the same sequence of 303 residues (see UniProt ID P77335). A list of native contacts for each structure was generated using the Contacts of Structural Units (CSU) software 25 . A native contact between residues i and j implies that at least one non-hydrogen atom from residue i and one from residue j are in contact in the native structure according to CSU. Contacts between residues within the modeled loops were deleted as these do not exist in the crystal structures of ClyA used here. The monomer has 735 native contacts, and the protomer has 612 native contacts. Both sets of native contacts are listed in the Supporting Information. Separate Cα SBMs for the monomer and protomer were generated using the above structures and their corresponding native contact maps through the SMOG Web server 23 . These two SBMs were integrated to create a Cα dual SBM of ClyA. For angles and dihedrals, we chose to define only those which differed in their native values between the two conformations by more than 18.12◦ and 54.50◦ respectively, to have dual potentials (Eq. 4 and 5-7). A deviation of 18.12◦ from a native angle corresponds to an energy penalty of 2, while a deviation of 54.50◦ from a native dihedral angle corresponds to an energy penalty of 1.4, on their respective single well potentials (Eq. 1). Using these cut-offs enabled us to use the minimal number of dual angle and dual dihedral potentials which could encode both conformations into a dual SBM. The remaining angles and dihedrals, as well as all of the bonds, were defined using single potentials (Eq. 1), and in practice, their parameters were taken from the Cα SBM of the monomer. For native contacts, those which were common 10 ACS Paragon Plus Environment


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to both conformations were represented by dual Gaussian contact potentials (Eq. 10). The interaction distances for the two Gaussian wells corresponded to native contact distances for that contact in the monomer and the protomer structures. Finally, native contacts unique to either conformation were defined using single Gaussian potentials (see Eq. 2) with interaction distances obtained from the respective conformations. See the following table (Table 1) for the number and the Supporting Information, for the lists of angles, dihedrals and contacts having single and dual potentials.

Numbers of various interactions potentials in the Cα dual SBM of ClyA Table 1: Numbers of angles, dihedrals and native contacts having single and dual interaction potentials

1 2

Interaction type Angle Dihedral

3

Native contacts

S.no

No. of single interaction potentials 268 251 411 (unique to the monomer), 288 (unique to the protomer)

No. of dual interaction potentials 33 49 324 (common to both monomer and protomer)

Coarse-grained membrane: membrane geometry and membrane-protein interactions We constructed a membrane made up of beads of unit mass. A set of 192 membrane beads were arranged in three layers with each layer having 64 beads. The inter-layer spacing was chosen to be 1.2 nm. Within each layer, the 64 membrane beads were arranged on a plane of 20 x 20 nm as an 8 x 8 grid with equal spacing between the beads (Fig. 3 shows the front view). The Z-coordinate of membrane beads present within a layer were position restrained so that they remain in the plane of their layer. However, they were allowed to move freely within the plane of their layer. All membrane beads were defined to have excluded volume 11 ACS Paragon Plus Environment

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interactions with an interaction distance of σN C = 1 nm (second term of Eq. 2). This three layer membrane was 4.4 nm thick, which is similar to the thickness of most biological membranes 26 . The interactions of the membrane with the Cα dual SBM of ClyA (see previous section) were defined as follows. Hydrophobic Cα atoms in the membrane-inserted segments (residues 1 to 35 and 177 to 203) were defined to favorably interact with the membrane beads through a 6-12 LJ potential having an interaction distance of 1.3 nm and an interaction strength of m (where, 0 < m < ). We found that for a fixed number of timesteps (Table 2, at a temperature of 0.9Tf for the Cα dual SBM of ClyA), m = 0.7 was the minimum interaction strength at which all replicates complete the conformational transition (see Discussion section “The fidelity of protomer formation might arise from helix αA1”). Hydrophobic Cα atoms were defined to be those corresponding to residues Ala, Ile, Leu, Tyr, Pro, Met, Trp, Phe, Cys, and Val 27 and are listed in the Supporting Information. Non-hydrophobic Cα atoms of the membrane-inserted segments, and Cα atoms of the extracellular segments were defined to have excluded volume interactions with the membrane beads with a σN C = 1 nm (second term of Eq. 2). The choice of having favorable membrane-protein interactions between the hydrophobic Cα atoms of the membrane-inserted segments in ClyA and the membrane atoms is elaborated in the first section of the Discussion. The simplicity of the membrane model is justified in the last section of the Discussion. In the simulation box of size 20 nm × 20 nm × 40 nm, ClyA was required to diffuse from its initial position in order to interact with the membrane. Since the center of mass of the system is kept fixed during the simulation, ClyA could not diffuse along the Z-axis as the membrane beads were constrained from moving along the Z-axis. We addressed this issue by adding a fictitious atom which had the same mass as that of the whole protein, but which did not have any interactions with either the protein or the membrane. The movement of this fictitious atom compensated for the motion of ClyA from its initial position. We additionally constrained this fictitious atom to move only along the Z-axis to capture the



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encounter between the membrane and the protein in a computationally reasonable time.

Figure 3: Front view of the initial snapshot of the coarse-grained membrane and the ClyA monomer showing the membrane beads as transparent spheres arranged in three stacks above the protein. The Cα atoms of the monomer are shown in as spheres colored blue (residues 1 to 35), green (residues 177 to 203), red (residues 268 to 303) and grey (rest of the protein).

Simulation details All simulations were run in the NVT ensemble using the stochastic dynamics integrator, with a timestep of 0.5 fs and a temperature coupling constant of 1 ps, using a modified version of GROMACS 4.5.4 in which the Gaussian contact potentials have already been implemented 22 . Electrostatic interactions were absent, van der Waal interactions were cut-off at 3 nm, and three dimensional periodic boundary conditions were used. As described in the Results section the choice of simulation temperature depends on the Tf , which is the temperature at which both folded and unfolded states are equally populated. 13 ACS Paragon Plus Environment

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We obtained the Tf of the Cα dual SBM of ClyA by performing simulations in the absence of the membrane at different temperatures (Table 2), found Tf to be 1.0974 (in reduced units, where 1 K = 0.008314 reduced units). At temperatures higher than the simulation temperature (0.9Tf < temperature < Tf ), we find protomer-like partially unfolded states (P*, Fig. S3) in which the β-tongue is unfolded and helix αA1 has separated from the rest of the tail domain but does not dock onto the head domain. However, the stable protomer is not observed in the absence of the membrane. For simulations in the absence of the membrane, 100 starting structures of the monomer were generated from a 2×107 timestep simulation of the Cα dual SBM at a temperature of 0.9561 (reduced units), while saving structures every 2×105 steps. These were used to initiate replicate simulations for 1.5×108 timesteps at the different temperatures mentioned in Table 2. Similarly we generated 100 starting structures of the protomer for the dual SBM simulations, from the single SBM of the protomer using the same parameters as above. These were used to initiate replicate simulations for 2×107 timesteps as mentioned in Table 2. For simulations in the presence of the membrane, the monomer was placed such that all of its atoms were at least 3.5 nm away from the closest membrane bead, and 100 starting structures were generated from 4×106 timestep simulations at 10 temperatures from 0.8397 to 0.9145 (in reduced units), while saving structures every 4×105 steps. The orientation and the position of the 100 starting structures is shown in Fig. S1. These were used to initiate 10 replicates (using only the first 10 starting structures) with different values of m between 0 and (see Table2) for 1.5×108 timesteps. At m = 0.7, 90 additional replicates (using the remaining starting structures) were carried out for 1.5×108 timesteps. In all, we obtained 100 replicate simulations which sampled the monomer to protomer transition at m = 0.7. It should be noted that these simulations are kinetic simulations and not equilibrium simulations performed at a transition temperature 8 or at a specific interaction strength 11 where back and forth transitions are observed between the folded (closed) and the unfolded



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(open) ensembles. In such kinetic simulations, less stable and rare conformations (such as the protomer conformation in the dual SBM simulations without the membrane and which are initiated from the monomer structure) can become accessible at longer timescales. Table 2: List of simulations Topology

Starting conformation

Temperature (reduced units)

Nreplicates

dual SBM

Monomer

0.9893

100

dual SBM

Monomer

1.0392

100

dual SBM

Monomer

1.0974

10

dual SBM

Protomer

0.9893

100

Monomer

0.9893

10

1.5×108 timesteps

Monomer

0.9893

+90

1.5×108 timesteps

dual SBM in the presence of the membrane with m = 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.57, 0.58, 0.6, 0.62, 0.65, 0.7, 0.75, 0.8 dual SBM in the presence of the membrane with m = 0.7

Nsteps 1.5×108 timesteps 1.5×108 timesteps 3×108 timesteps 2×107 timesteps

Analysis to obtain the negative logarithm of two dimensional probability distributions Reaction coordinates chosen to monitor the membrane-triggered conformational transition of ClyA were of two types. One category monitored the structural changes within the protein by quantifying the number of native contacts formed in different regions of ClyA. These regions and their native contact sets are defined in Results (see “Majority of the protein structural transformation occurs during the conversion from Im to the protomer”) and Fig. S11. We computed the extent of native contact formation only for contacts represented by single Gaussian potentials (i.e. native contacts unique to the monomer or the protomer) by using 15 ACS Paragon Plus Environment

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a continuous function as shown in Fig. 2D. If the distance between the Cα atoms forming a native contact is less than or equal to the native contact distance, it contributes +1 to the number of native contacts formed, or else it contributes as much as the negative of the Gaussian potential. This potential decays to zero as the distance between the Cα atoms increases beyond the native contact distance. For each of the five regions of ClyA (see Results and Fig. S11), the difference between the number of protomer specific (contacts unique to the protomer) and the number of monomer specific (contacts unique to the monomer) was obtained. This difference was normalized using the corresponding numbers of native contacts defined in the potential energy function of the Cα dual SBM of ClyA such that a value of 0 represents the monomer and a value of 1 represents the protomer. Additionally, distances between Cα atoms of residues 56 and 252, and that between 56 and 193 were also computed to capture the structural changes in the protein. The second category of reaction coordinates monitors different interactions between the membrane and the protein. These reaction coordinates were chosen to be (A) the effective number of contacts between (i) the protein and membrane, (ii) β-tongue and membrane, (iii) helix αA1 and the membrane, and (B) the number of residues of the membrane-inserted segments of ClyA which are inserted into the membrane. The effective number of contacts is defined as the interaction energy between segments of the protein and the membrane normalized by the strength of the membrane-protein interaction m used in that simulation. Contacts calculated in this way are denoted by Qm . The number of residues of the membraneinserted segments of ClyA which are inserted into the membrane are calculated by counting the number of such residues which are within the 4.4 nm envelope of the membrane. For several pairs of reaction coordinates (see Results), we plotted the negative logarithm of their two dimensional probability distributions, similar to previous studies 14,28 . Values close to 0 indicate a high probability of occurrence whereas higher values indicate a lower probability of occurrence. We also estimated the time of complete insertion of the β-tongue and helix αA1 into the



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membrane by identifying the timestep at which the Z-coordinate of the center of masses of the protein segment and membrane are within 0.5 nm of each other.

Error analysis for quantities computed from simulations The error in estimating any quantity (ξ) from a finite number of simulations can be obtained by using the Jackknife resampling method 29,30 . If there are n independent simulations that have been carried out, and if the value of the estimated quantity, ξ, from these n simulations is denoted as ξn , then we obtain the n partial estimates of this quantity (denoted by i ) from n − 1 simulations by omitting the data from the ith simulation. The following ξn−1

equation provides the variance associated with estimating the quantity ξ from n independent simulations using the partial estimates. n

n−1X i i V ar(ξn ) = (ξ − hξn−1 i)2 n i=1 n−1

(13)

If n is large, and if all simulations have sufficient sampling, the variance associated with omitting one simulation at a time will decrease to zero. We plot the square root of this variance as the error in estimating the corresponding quantity from our simulations, and find that the number of replicates chosen (listed in Table2) are sufficient to provide good estimates for these quantities. Errors corresponding to probability distributions shown in the article are plotted as separate figures in the Supporting Information. For the remaining quantities, errors are either shown as error bars on the figures or using the ± symbol in the text.

Solvent accessible surface area (SASA) calculations SASA calculations for the structure of the monomer (PDB 1QOY) and the protomer (PDB 2WCD, chain A) was performed only for residues 8 to 292. In addition, the CG1 and CG2 atoms of Valine 187 in 1QOY were deleted to mutate it into Alanine 187 so that the


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amino-acid sequence is the same for both conformations (see UniProt ID P77335). Using these structures, residue-wise SASA was calculated for the monomer and protomer using PSA software with a probe radius of 1.4 A 31 . Specifically, we defined hydrophobic SASA to be the SASA of the non-polar atoms (i.e. carbon atoms) from side-chains of hydrophobic residues Ala, Ile, Leu, Tyr, Pro, Met, Trp, Phe, Cys, and Val. Fractional SASA values were obtained by dividing the sum of the hydrophobic SASA of a set of residues by the total hydrophobic SASA of these same residues if they were in an Ala-X-Ala tripeptide. The latter is widely considered to be a model of the unfolded state for residue X 31 .



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Results The monomer to protomer conformational transition of ClyA is not observed in the absence of a membrane We constructed a Cα dual SBM of ClyA that encodes the structures of the monomer (PDB 1QOY) and the protomer (PDB 2WCD, chain A). The equilibrium values for bonded (bonds, angles and dihedrals) and non-bonded (native contacts) interactions in the dual SBM were derived from both structures. For example, native contacts unique to the monomer (monomer specific contacts) or the protomer (protomer specific contacts) were represented by single-well contact potentials, while those present in both structures and potentially having different interaction distances were represented using double-well potentials. The strength of all native contacts is the same as the energy scale of the potential energy function (, see Methods). We performed MD simulations of this dual SBM beginning with the monomer conformation. The simulation temperature was chosen based on the operating temperatures of most mesophilic proteins, which are observed to be ∼0.9-0.95 times the melting temperatures of the respective proteins 32 . In SBMs, the melting temperature (Tf ) is defined as the temperature at which the folded and the unfolded states are equally populated. Simulations of the dual SBM at 0.9Tf were analyzed by calculating the fraction of monomer specific (QM ono ) and protomer specific (QP roto ) native contacts formed. A given native contact is completely formed and has a value of one when the beads that are in contact are at or below their interaction distance in the crystal structures. This value decreases monotonically to zero as the two beads move further apart. The fraction of native contacts formed is then a sum of the extent of formation of individual native contacts normalized by the total number of native contacts. This definition is also applicable to a subset of native contacts. Fig. 4A shows the probability distribution of QP roto vs. QM ono (obtained from 100 independent replicate simulations). In this plot, the negative logarithm of the probability is shown along the two dimensions. Values close to 0 indicate a high probability of occurrence 19 ACS Paragon Plus Environment

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whereas higher values indicate a lower probability of occurrence. From Fig. 4A, we observe that only the monomer (M) is populated while the protomer (P) is inaccessible. We note that at a higher temperature of 0.95Tf , the monomer to protomer transition is observed, but it populates a partially structured protomer-like state in which the conformational transition is complete but, the membrane-inserting segments remain unstructured. (P*, Fig. S3). Moreover, since smFRET experiments find that the monomer is the predominant state in the absence of a membrane or detergent 6 , we chose to carry out further analysis at 0.9Tf where this is the case. In the next section, we develop a coarse-grained membrane model that allows ClyA to undergo its conformational transition at 0.9Tf .

Membrane interactions facilitate the formation of the protomer The ClyA pore structure suggests that the protomer has significant stabilizing interactions with the membrane 3 . We captured these interactions by constructing a coarse-grained membrane model consisting of three closely spaced layers of atoms. These atoms can diffuse freely only in their respective layers and have excluded volume interactions with each other (see Methods). We defined attractive interactions between the hydrophobic Cα atoms of the two membrane-inserted segments 3 (helix αA1: residues 1-35 and β-tongue: residues 177203, Fig. 1B) and the membrane particles using Lennard Jones (LJ) potentials. Each LJ potential defines a contact, with a minimum set at a distance of 1.3 nm, and with an interaction strength (m ) of 0.7. We choose m ∼ , as is the interaction strength for an intra-protein native contact in the dual SBM. Though helix αA1 contains charged residues, we haven’t explicitly included electrostatics or a desolvation penalty in our membrane model. This approximation may be reasonable because a separate study (Desikan et al., submitted) has shown through fully atomistic simulations that burial of the charged face of the amphipathic helix αA1 spontaneously creates a transmembrane water channel through the membrane which solvates the charged residues and thus reduces the desolvation penalty. This complex membrane-water system can thus be effectively described as a slab of attractive 20 ACS Paragon Plus Environment


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particles for the protomer. Our membrane model is conceptually analogous to the all-atom, highly-mobile-membrane-mimetic model 33 which expedites insertion of peripheral membrane proteins by mimicking the hydrophobic membrane interior using an organic solvent. We performed 100 independent MD simulations of the dual SBM at 0.9Tf , beginning with different orientations and positions of the monomer relative to the membrane (shown in Fig. S1, see Methods). All 100 replicates were simulated long enough to observe the formation of a membrane inserted protomer. The aggregate data from 100 transitions was used to plot the probability distribution of QP roto vs. QM ono (Fig. 4B) which shows that the protomer is accessible in the presence of the membrane. In addition, we also find that the formation of the protomer is irreversible. Fig. S4 shows that as the simulation proceeds, 100 % of the monomer population completely converts into the protomer conformation. Our results are consistent with single molecule Förster resonance energy transfer (smFRET) experiments which show that the monomer is the predominant state in the absence of detergent, and that the irreversible formation of the protomer requires addition of detergent 6 or membrane 34 . We note in passing that the irreversibility would be influenced by oligomerization of the membrane inserted protomers, but this effect is not considered in our model. Recent smFRET experiments 6 are able to grossly capture multiple steps in pore formation, the first of which is the conversion of the monomer into a single protomer. Our goal is to employ MD simulations of the dual SBM of ClyA to capture the structural details of this very first step at molecular resolution. Next, we investigate why protomer formation requires the presence of a membrane (or detergent). The protomer has fewer intra-protein native contacts as compared to the monomer. In absence of the membrane, the protomer to monomer transition is favorable at 0.9Tf (Fig. S5) suggesting that the protomer is less stable than the monomer. On the other hand, the monomer to protomer transition involves loss of intra-protein native contacts and is not favorable at 0.9Tf (Fig. 4A). This loss of contacts is more than compensated by favorable interactions with the membrane (Fig. S6). Therefore, favorable interactions between


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the protein and the membrane stabilize the protomer relative to the monomer and facilitate protomer formation. In subsequent sections, we report the mechanism, conformational landscape, and structural features obtained from the aforementioned 100 transitions of the monomer to the membrane inserted protomer at 0.9Tf .

Figure 4: Protomer is accessible only in the presence of the membrane. A) The negative logarithm of the two-dimensional probability distribution of the fraction of protomer specific contacts formed (QP roto ) vs. the fraction of monomer specific contacts formed (QM ono ) at 0.9Tf in the absence of the membrane shows that the protomer is inaccessible. The monomer and protomer states are marked by M and P respectively. B) Probability distribution at 0.9Tf in the presence of the membrane shows the presence of the protomer. Since these are kinetic simulations (see Methods), the population of M will reduce for longer simulations. The data was obtained from 100 independent replicate simulations for each of the two probability distributions plotted above. All of the replicate simulations were begun with the monomer as the starting conformation of the protein. The error in estimating the -ln(Probability) for both is shown in Fig. S2A, B.

A molecular description of the membrane-triggered conformational transition of ClyA In our simulations, the monomer freely diffuses in the simulation box until it is within interaction distance of the membrane. The conformational transition is subsequently initiated when either of the two membrane-inserting segments encounters the membrane, and we observe two pathways (Fig. 5). 22 ACS Paragon Plus Environment


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In pathway I, which occurs in 80 ± 4 % of the sampled trajectories, the β-tongue encounters the membrane and inserts as a helix-turn-helix motif (intermediate Im1 ). In Im1 , the head domain (of which a part comprises the β-tongue) transforms into its protomer conformation, while the middle and tail domains (see Fig. 1 for domain definitions) remain in their respective monomer conformations. From Im1 , binding of the N-terminus of helix αA1 to the membrane leads to the formation of intermediate Im2 . Im2 and Im1 can inter-convert between each other, i.e. the N-terminus of helix αA1 experiences multiple unbinding (Im1 ) and binding (Im2 ) events with the membrane. Im2 finally converts into the protomer when helix αA1 separates from the rest of the tail domain to fully insert into the membrane. This allows the rest of the tail domain and the middle domain to reorganize their helix interfaces to form the protomer. Since helix αA1 does not separate from the rest of the tail domain in the absence of the membrane (Fig. 4A), the membrane facilitates protomer formation (Fig. 5). In pathway II, which occurs in 20 ± 4 % of the sampled trajectories, the N-terminus of helix αA1 first binds with the membrane, followed by the insertion of the β-tongue as a helix-turn-helix motif. This results in the direct formation of Im2 which doesn’t revert back to only the helix αA1 bound state (Fig.5). As seen in pathway I, Im2 and Im1 inter-convert between each other. Finally, Im2 converts into the protomer. The primary difference between the two pathways is whether Im1 is on-pathway (pathway I; Im1 is formed directly) or offpathway (pathway II; the formation of Im2 can lead to the formation of Im1 ) to protomer formation.

The conformational landscape of the membrane-triggered conformational transition of ClyA In this section, we analyze the conformational landscape by plotting the probability distribution of the effective number of contacts between helix αA1 (αA1) and the membrane (Qm (αA1-mem)), vs. the effective number of contacts between the β-tongue (βT) and the 23 ACS Paragon Plus Environment

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Figure 5: The sequence of events occurring in the two pathways of the conformational transition of ClyA are illustrated. Helix αA1, β-tongue and helix αG (residues 268-303) are colored cyan, green and red respectively. The rest of the protein, and membrane particles are in grey. Schematics of pathways I and II and their fluxes are marked. These were inferred from analyzing 100 independent replicates of the dual SBM with the membrane, at 0.9Tf , with m = 0.7.



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membrane (Qm (βT-mem)) (Fig. 6A). The effective number of contacts is defined as the interaction energy between a membrane-inserted segment (αA1 or βT) and the membrane (mem) normalized by the strength of the membrane-protein interaction m . Contacts calculated in this way are denoted by Qm . In Fig. 6A, the population at (x,y) = (0,0) is that of the unbound monomer. We define a structural ensemble to be an intermediate if it undergoes a discernible intra-protein structural transformation and has a distinct population in the probability distribution. In pathway I (scheme shown in Fig. 5) the unbound monomer converts into the population at (x,y)∼(75,0) (observed in 80 ± 4 % of the trajectories). In this population (intermediate Im1 ), the β-tongue is inserted into the membrane as a helixturn-helix motif (Fig. 5). The population at (x,y)∼(75,20) corresponds to intermediate Im2 , in which the N-terminus of helix αA1 additionally interacts with the membrane but does not insert into it (see Fig. 5). Im1 and Im2 can inter-convert between each other, but together they represent a distinct structural ensemble (Im ) in which the β-tongue is inserted as a helix-turn-helix motif. All of the contacts that the β-tongue would form with the membrane in the protomer (i.e. x∼75) are already present in Im . Subsequently, the population at Im2 converts irreversibly into the protomer when a critical number of hydrophobic residues of helix αA1 (the first 12, see Fig. S8) insert into the membrane by thermal fluctuations. This leads to the complete insertion of helix αA1 into the membrane (see Fig. 5) and it forms all of its contacts with the membrane (population at (x,y)∼(75,65)). In pathway II (scheme shown in Fig. 5 and seen in Fig. 6A), the unbound monomer converts into the population at (x,y)∼(0,20) in which only the N-terminus of helix αA1 binds, but does not insert into the membrane. This population converts directly into Im2 and is observed in 20 ± 4 % of the trajectories. Im2 can inter-convert with the now offpathway Im1 , but eventually converts into the protomer. The observed flux in conformations suggests that pathway I is the major pathway. The difference in the flux between pathways I and II arises because encounters of the β-tongue with the membrane always leads to pathway I, while encounters of the N-terminus of helix αA1 with the membrane often lead back to


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the unbound monomer instead of pathway II. Despite the presence of two distinct pathways, we identify a consensus feature of the conformational transition. The distribution of the ratio of the time taken for helix αA1 to completely insert into the membrane, to the total time taken for the β-tongue to insert into the membrane (Fig. 6B) shows that this ratio is always greater than 1. Therefore, irrespective of which pathway is followed the β-tongue always inserts completely into the membrane before the complete insertion of helix αA1.

Majority of the protein structural transformation occurs during the conversion from Im to the protomer We next characterize the conformational landscape in terms of the structural transformations that occur within the protein. We defined five region-wise structural reaction coordinates in ClyA based on the difference between the number of protomer specific contacts and the number of monomer specific contacts formed (QP roto−M ono (region), see Figs. 7A,B and Fig. S11). The values of these reaction coordinates are normalized to lie between 0 (represents monomer) and 1 (represents protomer). Probability distributions of region-wise structural reaction coordinates versus the effective number of contacts between the protein and the membrane (Qm (Prot-mem)) are shown in Figs. 7C,D and Fig. S10. In these plots, the unbound monomer appears at Qm (Prot-mem) = 0, and the bound monomer in which only the N-terminus of helix αA1 interacts with the membrane (but is not inserted; Pathway II) appears at Qm (Prot-mem)∼10. In addition, Im1 and Im2 appear collectively as Im . We find that the head domain (residues 156-215, of which the β-tongue is a part) can sample its monomer and protomer conformations in both the unbound and N-terminus bound monomer states (Fig. 7C). Further, the head domain can either insert in its monomer conformation and then transform into its protomer conformation or, insert directly in its protomer conformation. We find that the latter is ∼10 times more probable than the former. However, their relative probability is a thermal effect i.e. at lower temperatures, the head domain is 26 ACS Paragon Plus Environment


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Figure 6: A) Probability distribution of the number of contacts formed between helix αA1 and the membrane (Qm (αA1-mem)) vs. the number of contacts formed between the β-tongue and the membrane (Qm (βT-mem)) shows the populations of the monomer (M, marked by a vertical ellipse), intermediates Im1 and Im2 which constitute the Im ensemble together, and the protomer (P). The flux between pathways (see Fig. 5) I (80 ± 4 %) and II (20 ± 4 %) was calculated separately. The error in estimating the -ln(Probability) for this probability distribution is shown in Fig. S7. B) The distribution of the ratio of the time of insertion of helix αA1 to the time of insertion of the β-tongue shows that this ratio is always greater than 1. The error bars show the error in estimating the frequency of occurrence for each value of the ratio of insertion times. Data for both A) and B) was obtained from 100 independent replicates.


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more likely to be in its monomer conformation at the time of insertion. Nevertheless, both types of insertion lead to the intermediate Im , in which the head domain is in its protomer conformation (Fig. 7C). Therefore, non-specific interactions between hydrophobic residues and the membrane stabilize the head domain in its protomer specific conformation. In contrast, helix αA1 samples only its monomer conformation in the unbound, and Nterminus bound (but not inserted) and intermediate (Im ) ensembles (Fig. 7D). The middle domain (Fig. S10 A,C) and the rest of the tail domain (i.e. apart from helix αA1, Fig. S10 B) also exhibit similar behavior. Therefore, only the head domain (i.e. ∼20% of the ClyA sequence) is in its protomer conformation in Im , while the rest of ClyA is still in its monomer conformation. This shows that in Im , ClyA has yet to undergo significant intra-protein structural transformations despite the complete insertion of one of the two membrane-inserted segments. In the final step, when Im converts into the protomer, all of the middle and tail domains including helix αA1 transform into their protomer conformations, and this constitutes a majority of the intra-protein structural transformation (Figs. 7D and Fig. S10).

A comparison of distances calculated from simulations with those derived from smFRET experiments. The kinetics of detergent facilitated conformational transition of ClyA was monitored using smFRET 6 . These experiments were initiated by mixing detergent with the labeled monomer (donor at residue 56, acceptor at residue 252). It was found that the monomer converts directly into the protomer. Our simulations captured the molecular mechanism of this direct pathway in the presence of a membrane. For comparison with this experiment, we plotted the probability distribution of the distance between the Cα atoms of residues 56 and 252 vs. Qm (Prot-mem) (Fig. 7E). The monomer and protomer are observed to have mean distances close to 5.5 nm and 4.45 nm respectively in agreement with their x-ray crystal structures and those inferred from the experimentally measured mean FRET efficiencies 6,35 . This suggests that the ensemble of structures corresponding to the monomer and the protomer in the FRET 28 ACS Paragon Plus Environment


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Figure 7: Structures of A) monomer and B) protomer. Colors correspond to five structural regions which are defined to have region-wise structural reaction coordinates (see Fig. S11 and Methods). These regions correspond to the head domain (blue), the middle domain (helix αA2 in purple and the rest of the middle domain, termed as mid-3α, in yellow), and the tail domain (helix αA1 in cyan and the rest of the tail domain, termed as tail-4α, in orange). The locations of experimental and proposed FRET probes are marked at residue positions 56, 193, and 252 (red stars) on both structures. C-F) Probability distribution of various reaction coordinates vs. the effective number of contacts between the protein and the membrane (Qm (Prot-mem)). These reaction coordinates are C) head domain (QP roto−M ono (head)), D) helix αA1 (QP roto−M ono (αA1)), E) distance between residues 56 and 252 (experimental FRET pair 6 ), and F) distance between residues 56 and 193 (FRET pair proposed here). In C, the two arrows represent insertion of the head domain in its monomer conformation (lower arrow), and in its pre-formed protomer conformation (upper arrow). In both E and F, dashed grey lines represent the native distances obtained from the structures of the monomer and the protomer. The error in estimating the -ln(Probability) for all the four probability distributions are shown in Fig. S9. Data for C-F) was obtained from 100 independent replicates.


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experiment are much like that observed in their respective x-ray crystal structures. This is important because the dual SBM employed here encodes the two x-ray crystal structures as the two end states of the conformational transition. Therefore, the end states in our simulations match those observed in the smFRET experiments. Interestingly however, we find that the intermediate Im also has a mean distance similar to that of the monomer (Fig. 7E). Therefore, it is possible that the intermediate Im was not distinguished from the monomer due to the choice of the FRET pair and hence was not detected. From our simulations, we find that the distance between residues 56 and 193 can distinguish between the monomer and Im , though not between Im and the protomer (Fig. 7F). We suggest that an additional monitoring of this FRET distance i.e. a probe which includes a FRET dye on the β-tongue, may be useful to identify the intermediate Im . Several mutations in the β-tongue (of which residue 193 is a part) abrogate lysis activity 2,5,36,37 . However the detection of Im will require labeling a residue in the β-tongue, and among all available alternatives we chose to suggest position 193 as its probability distribution (Fig. 7F) was able to best resolve the monomer from Im . Further, to the best of our knowledge, there are no reports of a single residue mutation at Isoleucine 193 being deleterious to ClyA’s lytic activity.

Discussion The ClyA dual SBM captures essential features of the conformational transition ClyA is a PFT that permeabilizes a target cell by forming non-selective pores 1,3,4,7 . The first step in this process is the transformation of ClyA from a soluble monomer (Fig. 1A) to the membrane inserted protomer (Fig. 1B). This conformational transition involves the conversion of a β hairpin into a membrane inserted helix-turn-helix motif, separation of helices αA1 and αA2, reorganization of the hydrophobic inter-helix interfaces, insertion of helix 30 ACS Paragon Plus Environment


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αA1 into the membrane and re-annealing of helix αA2 onto the rest of the extra-cellular segments 3,6 (Fig. 1). The exact sequence of these events is not known in molecular detail. We hypothesized that the underlying mechanism of this transition is built into the sequence and structures of ClyA, and constructed a dual SBM to capture this conformational transition. However, unlike the conformational transitions of soluble proteins 10–13 , a standalone dual SBM was insufficient to capture the conformational transition of ClyA (Fig. 4A). This was in agreement with a similar experimental observation 6 where the protomer was not observed in the absence of the membrane or detergent. It was necessary to construct a coarse-grained membrane in order to trigger the conformational transition because the protomer is less stable than the monomer and is stabilized by compensatory membrane-protein interactions (Fig. S6). The dual SBM with the membrane captures the molecular details of the conformational transition of the direct pathway 6 of monomer to protomer conversion (Fig. 5). It should be noted that the coarse-grained membrane has attractive interactions only with the hydrophobic residues of the membrane-inserted segments of ClyA. We support this choice by demonstrating that significant hydrophobic solvent accessible surface area (SASA) is buried into the membrane upon protomer formation. We find that the hydrophobic SASA (calculated from all-atom x-ray crystal structures PDB 1QOY and 2WCD, see Methods) for the membrane-inserted segments is 990 Å2 and ∼0 Å2 in the monomer and the membrane inserted protomer respectively, resulting in a burial of 990 Å2 . Similarly, for the extracellular segments, the hydrophobic SASA is 2453 Å2 and 2965 Å2 in the monomer and protomer respectively, resulting in an exposure of 512 Å2 . Taken together, this results in a net burial of 478 Å2 of hydrophobic SASA into the membrane. The experimentally determined transfer free energy 27,38 of 96 J/mol-Å2 for burial of hydrophobic SASA from solvent into a hydrophobic environment (either membrane or protein cores) implies that the burial of 478 Å2 of hydrophobic SASA gives a favorable thermodynamic bias of ∼46 kJ/mol (or ∼18 kB T at 310 K) for the membrane insertion of ClyA. Therefore, formation of the membrane


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inserted protomer involves burial of significant additional hydrophobic SASA into the membrane. This is in agreement with in silico estimates of the binding free energy of the ClyA protomer with a membrane (obtained from umbrella sampling simulations using the Martini force field with polarizable water and full electrostatics) of 55.6 ± 1.6 kJ/mol (Desikan et al., submitted). We argue further that our choice of defining favorable interactions only between hydrophobic residues of the membrane-inserted segments of ClyA and the membrane is reasonable because it achieves two additional objectives. First, it satisfies a key tenet of the structure-based model approach i.e. only interactions present in the experimentally determined structures are favorable and not others 9 . If future studies reveal the presence of alternative pathways for membrane insertion and conformational transition, additional intra-protein or membrane-protein interactions can be incorporated into the model to capture these pathways. Second, the hydrophilic residues of helix αA1 are defined to have excluded volume interactions with the membrane and these interactions are essentially repulsive in nature. This is conceptually similar to results reported in a separate study where a single membrane inserted protomer causes the formation of a transmembrane water channel in fully atomistic simulations due to the burial of charged residues within the membrane (Desikan et al., submitted). Therefore, despite the absence of electrostatic and desolvation effects, the amphipathic nature of helix αA1 is captured in the model with its hydrophobic and hydrophilic residues having favorable and repulsive interactions with the membrane respectively.

Partial absolute contact orders of the membrane-inserted segments can explain the mechanism of conformational transition The mechanism of conformational transition reveals that irrespective of which membraneinserted segment interacts with the membrane first, the insertion of the β-tongue and its structural transformation to a helix-turn-helix motif always precedes the insertion of helix 32 ACS Paragon Plus Environment


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αA1 into the membrane (Fig. 6). We substantiate this mechanism by using structural aspects of the β-tongue and helix αA1 from the monomer structure. The absolute contact order (ACO) of a protein is defined as the average sequence separation of all its native contacts 39 . By analogy, we define partial absolute contact order (pACO) as the average sequence separation of all the contacts in a given part of the protein. Protein folding studies have shown that ACO correlates well with the height of the unfolding free energy barriers 40 . This suggests that contacts that have shorter sequence separations (and lower pACO) unfold faster than contacts that have longer sequence separations. The β-tongue has partially solvent-exposed hydrophobic residues as suggested by its fractional SASA of 0.4 (i.e. 40 % of its total hydrophobic surface area is solvent exposed, see Methods for SASA calculations). Its secondary and tertiary structural native contacts are low in number (80 contacts) with a low packing fraction of 3 contacts per residue, and a low partial absolute contact order (pACO) of 24 (Fig. 8). While low packing fraction implies a low density of contacts and therefore lower stability 41 , low pACO implies a low free energy barrier to disrupt those contacts 40 . Furthermore, of the total amount of hydrophobic SASA buried into the membrane (990 Å2 , see previous section), ∼86 % (i.e. 849 Å2 ) arises from the burial of the β-tongue alone. The β-tongue is stabilized primarily by interactions with the membrane even in the assembled pore 3 and the observed mechanism (Fig. 5) suggests that these stabilizing interactions are established early during the transition. Thus, the presence of flexible (low packing fraction, low pACO, high flexibility - see Fig. S12) and partially exposed hydrophobic residues on the β-tongue could be a useful design feature that enables ClyA to sense and favorably interact with membranes. In contrast to the β-tongue, we find that the hydrophobic side-chains in helix αA1 have a low fractional SASA of 0.09 (see Methods), and are an integral part of the hydrophobic interhelix interfaces in the tail domain. The secondary and tertiary structural native contacts in helix αA1 are larger in number (157 contacts), correspond to a larger packing fraction of 4.5, and have a larger pACO of 154 (Fig. 8). These characteristics suggest that helix


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αA1 is likely to be more stable and rigid relative to the β-tongue. The difference in pACO values of the two membrane-inserted segments suggests that helix αA1 experiences a larger free energy barrier while separating from the rest of the tail domain as compared to the free energy barrier experienced during the transformation of the β-tongue into a helix-turn-helix motif. This explains the reason behind the β-tongue inserting as a helix-turn-helix motif before the insertion of helix αA1 (Fig. 6). Further, we speculate based on the large pACO of helix αA1 that its separation from the tail domain makes the conformational transition rate-limiting to pore formation at low µM concentrations of the monomer and in the presence of detergent 6,34 , and predict that mutations which marginally destabilize the hydrophobic inter-helix interfaces in the tail domain are likely to accelerate pore formation under similar conditions. Unlike a membrane, free detergent may be unable to form a sufficiently large matrix that can separate helix αA1, and it likely drives the conformational transition by stabilizing conformations in which helix αA1 separates from the tail domain by thermal fluctuations. It is possible that membrane mediated separation of helix αA1 (Fig. 5) can accelerate this step, and underlies the ten-fold faster kinetics observed in experiments with erythrocytes 34,35 .

The fidelity of protomer formation might arise from helix αA1 Previous experiments have shown that the rates of cell lysis by ClyA vary for erythrocytes from different species 42 , suggesting that target cell membrane composition also determines lysis. For example, ClyA does not lyse bacterial outer-membrane vesicles which facilitate secretion of the monomer, possibly due to the absence of cholesterol in their membranes 1 . We find a cholesterol recognition amino acid consensus (CRAC) motif 43 of the kind L/VX1−5 -Y-X1−5 -R/K (where X is any residue) in helix αA1 (residues 24 to 29 - LDLYNK). Such motifs have been posited to be essential for cholesterol recognition and binding in numerous membrane proteins 43 . Since the presence of cholesterol in target membranes is a requirement for pore-formation by ClyA 1,5 , we speculate that its amount in the membrane represents the 34 ACS Paragon Plus Environment


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Figure 8: The partial absolute contact order (pACO) of the β-tongue in the monomer and protomer shows that it increases during the conformational transition. On the other hand, the pACO of helix αA1 is much larger in the monomer, and it decreases to about half its value when converting from the monomer to the protomer. The pACO values of the β-tongue, the helix αA1 and the ACO of the whole protein are markedly different from each other only in the monomer, but become similar in the protomer. These observations hold good even for pACOs computed from all-atom contacts suggesting that on average the contact densities appear to be similar irrespective of whether contacts are weighted according to the number of all-atom interactions or not (see Fig. S13).

biological manifestation of the membrane-protein interaction strength (m ) in our model and this parameter can tune the reliability with which ClyA can form protomers (see Methods). To examine the effect of m using our model, we plotted the average number of membrane inserted residues from the membrane-inserted segments vs. m (Fig. 9). When all the residues from the membrane-inserted segments are inserted, the conformational transition is complete. As expected, we see an increase in the average number of membrane-inserted residues with increasing values of m (Fig. 9). However, we find that there are two distinct transitions in this profile, one each corresponding to the insertion of the two membrane-inserted segments of ClyA. The transition at lower m (midpoint at m = 0.35) and higher m (midpoint at m = 0.58) correspond to the insertion of the β-tongue and helix αA1 into the membrane respectively. We reason that the lower kinetic barrier for the conformational conversion of the β-tongue as compared to that for helix αA1 causes the appearance of these two distinct transitions. As discussed earlier, the difference in the kinetic barriers for the conformational 35 ACS Paragon Plus Environment

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conversion of the β-tongue and helix αA1 also arise from differences in the pACO (Fig. 8) of these segments in the monomer. This provides a structural basis for the presence of the two distinct transitions in Fig. 9. The sequence of ClyA could have evolved so that interaction with bacterial membranes allows the insertion and dissociation of the β-tongue but not helix αA1, while target cell membranes lie at higher values of m (Fig. 9). If this is indeed the case, then the β-tongue acts as a quick response sensor for detecting the membrane, while helix αA1 acts as a fidelity check which ensures that ClyA inserts into specific membranes with high reproducibility and less in others. Previous experiments on ClyA suggest that deletions in helix αA1 do not affect binding to erythrocyte membranes but significantly attenuate pore formation and hemolytic activity 37 . These experiments, together with the presence of the CRAC motif in helix αA1 in ClyA, are in agreement with our findings that the β-tongue binds and inserts into membranes, while helix αA1 ultimately completes the conformational transition required for pore formation.

Comparison with a previously hypothesized mechanism of conformational transition of ClyA A previous study proposed a mechanism of conformational transition of ClyA by comparing the crystal structures of the monomer and the protomer 3 . It was suggested that F190 of the β-tongue forms a latch that locks two loaded springs into a cluster of aromatic residues formed by F50, Y54, F159, Y165, F190. Spring 1 partly consists of F159 and Y165, while F50 and Y54 are part of spring 2. When the monomer encounters the membrane, hinges at G180 and G201 are thought to facilitate swinging out of F190 and the β-tongue thus disrupting the F190 latch in the aromatic cluster and freeing springs 1 and 2. The swung out β-tongue inserts first into the membrane, and undergoes a partial structural transformation in which it along with helix αD merge with helix αC into a single helix due to the relaxation of spring 1. Next, helices αA1 and αA2 separate from the tail and middle domains respectively 36 ACS Paragon Plus Environment


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Figure 9: The average number of residues which insert into the membrane are shown as a function of the membrane-protein interaction strength (m ). A residue is considered to be inserted if it is within the 4.4 nm envelope of the membrane. The data points (dots) are obtained by averaging over the last 1000 timesteps taken from each of 10 independent replicates (total timesteps = 1.5×108 ) at 0.9 Tf at each m value. The error bars show the error in estimating the mean value. For some data points, error bars are not visible because they are smaller than the size of the data point. The data for helix αA1 (cyan) and the β-tongue (green) were fit to sigmoid curves. The residuals of the fits are shown in Fig. S14 with the same color scheme. The profile for the insertion of the total membrane-inserted segment (black) is the sum of the profiles of helix αA1 and the β-tongue, and it fits better than a single sigmoid (residual in grey, see Fig. S14). The lowest value of m at which all the residues from the membrane-inserted segments are inserted into the membrane (m = 0.7) was chosen to carry out simulations which were used to infer the conformational landscape of the membrane-triggered conformational transition of ClyA.


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(supposedly in a membrane-independent manner) due to the relaxation of spring 2. Helix αA1 being amphipathic binds to the membrane with its hydrophobic residues facing the interior of the membrane while helix αA2 docks onto helix αC. After the separation of helix αA1 from the tail domain, the remainder of the tail domain reorganizes itself to bury the hydrophobic residues of helices αB, αC, αF and αG which were earlier sheathed by helix αA1. Finally, helix αE and the N-terminus of helix αF reorganize to complete the membrane inserted helix-turn-helix motif in the head domain. However, MD simulations or experiments had not been carried out to validate this mechanism. The proposed mechanism and our simulations both suggest that the β-tongue inserts first into the membrane followed by helix αA1, and that the separation of helix αA1 is followed by the reorganization of the rest of the tail domain into its protomer conformation. However, we do not observe the proposed latching mechanism or the double-spring-loaded mechanism. Instead, our model suggests that the separation of helix αA1 from the rest of the tail domain is mediated by the membrane. We argue that the large pACO of the native contacts of helix αA1 (Fig. 8) in the monomer poses a large free energy barrier which may not be surmounted by the relaxation of a loop (i.e. spring 2) that does not interact with the tail domain. Therefore, the dominant structural feature that determines the separation of helix αA1 appears to be the large pACO of its native contacts and not the local relaxation of springs 1 and 2. Overall, the mechanism observed in our simulations fine-tunes the previously proposed mechanism by providing an energetic basis for the order of events.

A simple membrane model reveals the membrane-like features that are required for facilitating protomer formation Protomer formation and pore assembly by ClyA requires the presence of detergent or membrane 6,34 . Therefore, in addition to protein sequence and structure, a membrane or membranelike environment also determines conformational fate. By accounting for the membraneprotein interactions of the protomer in a reduced membrane model, we captured the monomer 38 ACS Paragon Plus Environment


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to protomer conformational transition. We also found that (i) the underlying mechanism arises due to the structural features of the monomer, and that (ii) membrane-like features stabilize the protomer. Therefore, as long as the membrane stabilizes the protomer, the dynamics of the conformational transition arises from well-established SBMs. A coarse grained membrane with simple energetics was able to tease out the influence of the membrane because of its simplified structure and energetics. Membrane complexity can be increased by adding structural and chemical features such as hydrophilicity of the membrane-water interface, lipid density and fluidity. A structurally and chemically more complex membrane model will possibly affect the energetics of protomer formation and specific protein-lipid interactions (as seen for other proteins 44,45 ). In addition, increasing the complexity of the protein by explicitly representing side-chains could also be used to test the strength of the springs in the latching mechanism proposed earlier 3 (see previous section). However, we do not expect the underlying mechanism to alter significantly from the one observed here, because the mechanism is built into the broad structural features 46 of ClyA (Fig. 8).

Conclusions We captured the membrane-triggered monomer to protomer conformational transition of E. coli ClyA, an α-helical PFT, using MD simulations of its dual SBM in the presence of a coarse-grained membrane. ClyA has two membrane-inserted segments, the β-tongue in the head domain and helix αA1 in the tail domain (Fig. 1). Favorable interactions between hydrophobic residues of these membrane-inserted segments and the membrane not only trigger the monomer to protomer transition, but also stabilize the relatively less stable protomer conformation. Even though the membrane facilitates protomer formation, we find that the mechanism of the conformational transition is built into the structure of the monomer. We compared the observed mechanism with recent smFRET experiments and suggest an


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additional FRET pair that can help to detect a partially inserted intermediate ensemble (Fig. 7E,F) in experiment. Overall, the observed mechanism (Figs. 5 and 6) and several experimental observations such as the rate-limiting nature of the conformational transition 6,34 , and variation of pore-formation rates with detergent and membrane sources 6,34,35,42 can be explained by the large pACO (the average sequence separation of a given set of native contacts) of helix αA1 in the monomer (Fig. 8). We expect that destabilizing mutations in the hydrophobic inter-helix interfaces of the tail domain are likely to accelerate the conformational transition. ClyA appears to be designed such that the less stable and more flexible β-tongue has partially exposed hydrophobic residues that are poised to insert into the membrane (a quick response sensor), while the more stable and less flexible helix αA1 with buried hydrophobic residues ensures the fidelity of protomer formation only in target cell membranes (Fig. 9). Unlike most PFTs which have only one membrane-inserted segment, these design features are likely to be unique to ClyA and other proteins in the ClyA family of PFTs. Nevertheless, acceleration of protomer formation by membrane-protein interactions is likely to be applicable to PFTs in general. We conclude that using a coarse-grained membrane in conjunction with dual SBMs can be a computationally inexpensive way to provide microscopic insights into membrane-triggered protein conformational transitions, and expect that such simulations will be a guide for future experiments on ClyA and other PFTs.

Supporting Information Available Supporting figures: Position and orientation distributions of ClyA prior to membrane interaction (Fig. S1), Error estimation of probability distributions of Fig. 4 (Fig. S2), Probability distribution similar to Fig. 4A, but at 0.95Tf (Fig. S3), Irreversibility of the conformational transition in the presence of the membrane (Fig. S4), Probability distribution of the protomer to monomer backward transition in the absence of the membrane (Fig. S5), Probability distribution of the intra-protein native contact energy vs. the effective number of contacts between the protein and the membrane (Fig. S6), Error estimation of probability 40 ACS Paragon Plus Environment


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distributions of Fig. 6A (Fig. S7), Probability distribution showing the number of residues of helix αA1 which are inserted into the membrane during the monomer to protomer transition (Fig. S8), Error estimation of probability distributions of Fig. 7C-F (Fig. S9), Probability distributions along the structural reaction coordinates from middle and tail domains of ClyA (Fig. S10), Native contact maps of monomer and protomer showing the various reaction coordinates (Fig. S11), Debye-Waller factors for the ClyA monomer computed from all-atom MD simulations (Fig. S12), Partial absolute contact order computed from the all-atom contact map (Fig. S13), Residuals of the fits shown in Fig. 9 (Fig. S14). Supporting text: List of native contacts used to construct the single SBMs of the monomer and protomer, List of native contacts, angles and dihedrals used to construct the dual SBM of ClyA, as mentioned in Table 1, and List of residue numbers of the membrane-inserted segments of ClyA which have LJ interactions with membranes.

This material is available free of charge via the Internet at http://pubs.acs.org/.

Acknowledgement SG thanks the Government of India, Department of Atomic Energy (Core funding), and Government of India, Department of Science and Technology - Ramanujan Fellowship (Grant no. SR/S2/RJN-63/2009, 5 years, wef 15/04/2010) for funding. RD and KGA thank the Department of Science and Technology (DST), Government of India, for a grant under which this work was carried out. All the authors thank Dr. Rahul Roy (Indian Institute of Science) for discussions.

Abbreviations PFT, pore forming toxin; ClyA, Cytolysin A; SBM, structure based model; dual SBM, dual structure based model; MD, molecular dynamics; pACO, partial absolute contact order; smFRET, single molecule Förster resonance energy transfer. 41 ACS Paragon Plus Environment

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