Tetrameric Charge-Zipper Assembly of the TisB Peptide in

Subscriber access provided by Iowa State University | Library

B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

Tetrameric Charge-Zipper Assembly of the TisB Peptide in Membranes - Computer Simulation and Experiment Violetta Schneider, Parvesh Wadhwani, Johannes Reichert, Jochen Bürck, Marcus Elstner, Anne S. Ulrich, and Tomas Kubar J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b12087 • Publication Date (Web): 01 Feb 2019 Downloaded from http://pubs.acs.org on February 3, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Tetrameric Charge-Zipper Assembly of the TisB Peptide in Membranes – Computer Simulation and Experiment Violetta Schneider,† Parvesh Wadhwani,‡ Johannes Reichert,‡ Jochen Bürck,‡ Marcus Elstner,† Anne S. Ulrich,‡ and Tomáˇs Kubaˇr∗,† Institute of Physical Chemistry, Karlsruhe Institute of Technology, 76131 Karlsruhe, Germany, Institute of Biological Interfaces (IBG-2), Karlsruhe Institute of Technology, 76021 Karlsruhe, Germany, Institute of Organic Chemistry, Karlsruhe Institute of Technology, 76131 Karlsruhe, Germany, and Center for Functional Nanostructures, Karlsruhe Institute of Technology, 76131 Karlsruhe, Germany E-mail: [email protected] Phone: +49 721 608 43574. Fax: +49 721 608 45710

∗

To whom correspondence should be addressed Institute of Physical Chemistry, Karlsruhe Institute of Technology, 76131 Karlsruhe, Germany ‡ Institute of Biological Interfaces (IBG-2), Karlsruhe Institute of Technology, 76021 Karlsruhe, Germany ¶ Institute of Organic Chemistry, Karlsruhe Institute of Technology, 76131 Karlsruhe, Germany § Center for Functional Nanostructures, Karlsruhe Institute of Technology, 76131 Karlsruhe, Germany †

1

ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract TisB is a short amphiphilic α-helical peptide from E. coli that induces a breakdown of the pH gradient across the inner membrane when the bacteria are under stress and require to form persister cells to turn into a biofilm. A computational–experimental approach combining all-atom and coarse-grained molecular dynamics simulation with circular dichroism spectroscopy and gel electrophoresis was used to reveal its structure and oligomeric assembly in a phospholipid bilayer. TisB is found to be inserted upright in the membrane as a tetrameric bundle with a left-handed sense of supercoiling, best described as an antiparallel dimer-of-dimers. The tetramer is stabilized by means of a regular but dynamically interchanging pattern of salt bridges and hydrogen bonds, in accordance with the recently proposed ‘charge zipper’ motif.

Introduction TisB is a 29-residue amphiphilic peptide (MNLVDIAILILKLIVAALQLLDAVLKYLK) that localizes to the inner membrane of E. coli and is the toxic component of one of the bacterial toxin–antitoxin systems. 1,2 TisB is responsible for temporarily suppressing metabolic processes and other important cellular functions such as transcription. To protect itself from environmental stresses, E. coli overproduces TisB, which binds to the inner membrane as an amphiphilic α-helix. It is able to insert into the lipid bilayer presumably as an oligomeric assembly, to break down the proton-motive force by equilibrating the pH gradient across the membrane. 2–4 This leads to a depletion of ATP levels in E. coli, thereby inducing a state of dormancy and a subsequent switch to a biofilm growth mode. The surviving persister cells are thereby able to exhibit resistance against further environmental stresses like antibiotics. 3 Thus, following this cascade of events, the stress-response peptide TisB is highly important for bacterial survival and plays a critical role in the formation of biofilms in which E. coli can survive. Biophysical functional studies of TisB in planar lipid bilayers showed that it forms anion2


Page 2 of 30

Page 3 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


selective channels, though with a small diameter, and it was proposed that the leakage of protons may actually proceed by way of a hydroxyl anion transfer in the opposite direction. 5 Circular dichroism (CD) and oriented CD studies indicated that TisB folds into a helical structure and aligns into a transmembrane state in lipid bilayers, with a Hill coefficient of two that suggested the minimal functional assembly of TisB to be a dimer. 6 Coarse-grained (CG) molecular dynamics (MD) simulations showed that TisB in a transmembrane state will spontaneously assemble into an antiparallel dimer. This was predicted to be stabilized by a ladder of salt bridges connecting the two transmembrane helices, a so-called ‘charge zipper’, 7 which avoids the exposure of charged residues in the hydrophobic interior of the bilayer. Fig. 1 illustrates how the strip of alternating charges on the otherwise hydrophobic TisB helix could traverse the lipid bilayer if it is assembled as an antiparallel dimer, 6 and the central Gln that could engage in hydrogen bonding. The concept of a charge zipper in an otherwise hydrophobic membrane environment 7 was first discovered in the putative pore-forming TatA complex of the twin-arginine translocase of E. coli, 8 and was recently reported also in the respiratory supercomplex factor 1 (Rcf1) from the inner mitochondrial membrane of S. cerevisiae. 9

Figure 1: TisB folds as an amphiphilic α-helix that carries a narrow strip of charged residues along one face, as illustrated by the helical mesh representation (yellow – hydrophobic, red – anionic, blue – cationic, light blue – polar). An antiparallel dimer could be immersed in a transmembrane alignment by forming four intermolecular salt bridges (purple: D5–K26, K12–D22, D22–K12, K26–D5) plus a hydrogen bond (green: Q19–Q19). The postulated ladder of salt bridges constitutes a so-called ‘charge zipper’ that shields the charges in the hydrophobic bilayer core.

3



The interaction of membrane-active peptides with lipid bilayers has been commonly investigated with MD simulations. 10–13 MD is a particularly useful approach that is complementary to experimental studies, because the relevant peptide-peptide and peptide-lipid interactions are not readily accessible with conventional methods like X-ray crystallography or liquid-state NMR, and atomic details are difficult to study by solid-state NMR. 14–16 Typical challenges with membrane-bound peptides are variable conformations and multiple alignment states in a lipid bilayer, as well as the ability to oligomerize and engage in highly dynamic equilibria. MD simulation – or even better, multi-scale schemes employing MD – may thus be the key to characterizing the interchanging structures of membrane-bound peptides, and thereby understanding their functional mechanisms. 17 Some of the general challenges with MD simulations, however, are due to potential incompatibilities of the force fields used for the lipids and peptides, as well as the lack of electronic polarizability of the most popular force fields. 18–20 Not less serious is the issue of undersampling in MD simulations, which occurs in even the simplest peptide–bilayer complexes due to the comparatively slow motions of the lipid tails. 21–23 This problem may be partially alleviated by passing to a CG representation, 24 like that in the Martini force field, 25,26 which leads to an acceleration of two to three orders of magnitude in the accessible sampling. Still, due to the slow lateral diffusion in lipid bilayers coupled to the long life times of nonoptimal dimers, free CG MD simulation cannot describe the whole process that consists of peptide insertion into a membrane and secondary structure adaptation, induced by the insertion into the membrane and, later, by the oligomerization of the peptide. This challenge can be met by an application of the recently developed docking assay for transmembrane components (DAFT). 27 The purpose of DAFT is to find preferred orientations of the individual peptide components within an oligomeric structure. This is accomplished by performing many different simulations in parallel, with each simulation starting from a different orientation of the individual peptide monomers. The resulting associated oligomers may be eventually analyzed with, e.g., clustering algorithms.

4


Page 4 of 30

Page 5 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


CG simulation are particularly useful in exploration of structures and orientations with low resolution. The loss of detailed atomic structure may hamper the use of CG simulations whenever any specific interactions or fine conformational changes play a role. 24 In such cases where detailed features are of interest, it is a promising strategy to couple CG and all-atom representations. Then, their respective advantages – large-scale sampling and more detailed description of relevant structural states – are combined. 12,28–30 A natural way to exploit this idea in the DAFT context is to consider the oligomeric structures identified by DAFT as starting geometries for all-atom MD simulations. The goal of this work is to resolve the structure of oligomeric TisB assembly and its orientation in a lipid bilayer. The structure and dynamics of the assembly was determined by means of DAFT and then refined with all-atom MD simulations, similarly to a recent work on a transmembrane protein. 31 Note that even though a Hill coefficient of two had been observed for TisB-induced leakage of vesicles, which indicates that dimers are involved, a higher-order oligomeric state has never been ruled out. Tetrameric or hexameric TisB would also be compatible, as long as they are based on a minimal cooperative unit of a dimer. Therefore, in order to understand how TisB assembles and functions in the membrane, this work aimed to characterize its oligomeric state, the relative alignment of the monomers in the bundle, the orientation of the entire assembly in the lipid bilayer, and to examine the possible charge zipper that had been predicted. Generally, the quality of results from biomolecular simulations is often sensitive to technical parameters like the choice of the force field, presence or absence of counterions, and the treatment of electrostatics. 32,33 A practicable way to verify the observations in MD simulations of membrane-bound peptides is to complement the study by comparison with the outcome of biophysical experiments. 13 Such an approach was exploited here as well, and the actual oligomeric state was assessed on the basis of experimental findings. Thus, the appearance of a complex charge zipper in TisB was characterized and discussed.

5



Methods Initial structural model An α-helical model of the 29-residue monomer of TisB was constructed using the xLeap tool from the AmberTools package. 34 An α-helical conformation was achieved with the choice of backbone dihedral angles of ϕ = −57◦ and ψ = −47◦ . The termini of the peptide chains − were considered charged (NH+ 3 and COO , respectively). Since the termini will always be

located in the region of polar lipid head groups or even in the aqueous phase, their charges will be screened by such high-dielectric environment. Therefore, the terminal charges will hardly affect the interactions in the interior of the lipid bilayer.

Docking assay for transmembrane components in coarse-grained representation The DAFT procedure 27 was executed for TisB twice – first, starting from two separate TisB strands in an antiparallel arrangement (↑↓) to assemble into a dimer, and second, starting from four separate TisB strands (↑↓↑↓) as a basis for tetrameric structures to emerge. In each case, 400 simulations with different initial orientations of the monomers, separated by 3.5 nm, were performed within a DAFT run. The simulation times were 1 µs and 3 µs in the case of dimer and tetramer, respectively. All of the peptides were embedded in a pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer, which was solvated with water, and the system was electro-neutralized with an appropriate number of chloride ions. The choice of the lipid was made for consistence with the experimental part of the study, and the temperature of 300 K ensured the CG POPC bilayer to remain in the fluid phase. All of the system was described with the coarse-grained force field Martini 2.2. 35 The simulations of dimer and tetramer were performed according to the standard DAFT protocol, and details are given in the Supporting Information. Briefly, the initial helical model of TisB monomer was aligned along the z-axis first. This all-atom model was con6


Page 6 of 30

Page 7 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


verted to a coarse-grained representation, and then duplicated in an antiparallel orientation. The system size was ca. 3,000 beads and ca. 5,400 beads for TisB dimers and TisB tetramers, respectively. An equilibration procedure was performed for each of the 400 different configurations created as described above, for both the dimer and the tetramer of TisB. An energy minimization with the steepest-descents algorithm was followed by a constant-temperature (NVT) simulation of 10 ps and by a constant-temperature-and-pressure (NPT) simulation of 100 ps. Finally, a production simulation at 300 K and 1 bar was run. The total sampling amounted to 400 × 1 µs for TisB dimer, and to 400 × 3 µs for TisB tetramer.

Conversion to all-atom representation and re-embedding into lipid bilayer Clustering analysis was performed on the final structures from the DAFT-CG simulations, separately for the TisB dimer and the TisB tetramer. Then, the structures of two of the most populated clusters from DAFT, for each case, were selected for further processing. The CG structures were converted to an all-atom representation with the Martini tool Backward, 36 which uses a library of mapping definitions for the geometric reconstruction. The resulting structures were subsequently relaxed with energy minimization followed by short MD simulations, following the published standard protocol of Backward. Afterwards, the dimer/tetramer of TisB was aligned along the z-axis, which corresponds to the lipid bilayer normal. The oligomer was inserted into a pre-equilibrated fully hydrated POPC bilayer; that system consisted of 288 lipid molecules (144 per leaflet) and ca. 11,500 water molecules. That insertion was performed with the Gromacs tool Membed, 37 applying standard parameter values. With Membed, the TisB oligomer was first reduced in size in the membrane plane, and any overlapping lipids and water molecules were removed. Then, the original size of the peptide was restored, and the procedure was concluded by a short MD simulation. Finally, the system was electro-neutralized by addition of four chloride ions. The simulations of TisB dimer comprised ca. 72,700 atoms (2 TisB strands, 278 POPC 7



molecules, ca. 11,500 water molecules and 2 chloride ions); there were ca. 73,700 atoms in the simulations of TisB tetramer (4 TisB, 278 POPC, ca. 11,500 waters and 4 chlorides). TisB and POPC were described with AMBER14SB 38 and Slipids 39,40 force fields, respectively. The TIP3P water model was used, 41 as was the parametrization of ions by Joung & Cheatham. 42

All-atom simulations The all-atom system was first energy minimized with steepest-descents. Then, an NVT simulation at 308 K for 1 ns was performed, and was followed by an NPT simulation at 308 K and 1 bar for 1 ns. Both of these simulations involved harmonic position restraints on the non-hydrogen atoms of TisB. Finally, 1.01 µs of NPT simulation at 308 K and 1 bar was performed for data production; the initial interval of 10 ns was considered as the last stage of equilibration and was discarded prior to any analyses. The temperature of 308 K is in accordance with the conditions of solid-state NMR experiments, 16 and is high enough to keep the lipid in the fluid phase; note that there is no such concern in DAFT simulations, which were performed at a slightly lower temperature of 300 K. All simulations were performed using the molecular simulation package Gromacs 43–45 in the versions 4.6.7 (DAFT simulations) and 2016.3 (all-atom simulations). MD trajectories were analyzed with Gromacs 2018.3 and Plumed 2.4.3. 46 Molecular structures were visualized with VMD 1.9.2, 47 and numeric data were plotted with Grace 5.1.24. 48

Analysis Cluster analysis The DAFT procedure yielded 400 coarse-grained structures for each of the investigated oligomeric systems, and it was crucial to discover any frequently occurring patterns. To this end, a cluster analysis was performed on the ensembles of final structures from DAFT, using the Gromacs tool Cluster. Similarity of the individual structures was assessed by

8


Page 8 of 30

Page 9 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


means of root-mean-square deviations (RMSD) of the atomic coordinates used as a criterion in combination with a single linkage algorithm with a cut-off radius of 0.4 nm and 0.7 nm for TisB dimer and TisB tetramer, respectively. The two most populated structures were considered as starting structures for subsequent all-atom simulations. Helicity To calculate the helicity for each amino acid residue, the dihedral angles ϕ and ψ were obtained for every snapshot from the trajectory. A residue was considered helical as long as both ϕ and ψ were within 30◦ of their idealized values of ϕ = −57◦ , ψ = −47◦ . Then, the helicity of an amino-acid residue in a trajectory was obtained as the percentage of frames in which this criterion was met. Tilt angle Tilt angle is the angle between the helical axis of the peptide and the membrane normal direction, represented by the z-axis of the coordinate system in the simulations. The helical axis of the peptide was constructed as the straight line connecting the centers of mass of the N-and C-terminal segments of the molecule, represented by residues 1–14 and residues 16–29, respectively. The tilt angle was then obtained with the Gromacs tool Bundle. Solvent-accessible surface area (SASA) SASA was calculated for different TisB dimer assemblies to assess their compactness. The algorithm from Ref. 49 as implemented in the Gromacs tool Sasa was used.

Experimental methods Peptide synthesis and purification TisB was synthesized on an automated peptide synthesizer and purified as described previously. 6 The purified TisB was characterized using mass spectrometry. 9



CD spectroscopy For CD measurements, a stock solution of TisB with a concentration of 0.5 mg/ml was prepared. The spectrum of TisB was measured in 10mM SDS at 25 °C using methods described previously. 50,51 SDS PAGE The purified TisB was dissolved in SDS buffer, and polyacrylamide gel electrophoresis (SDSPAGE) was performed by standard protocols. 51,52

Results TisB dimer The structure and orientation of an antiparallel dimer assembly of TisB was first explored with the DAFT scheme based on CG simulations. Clustering analysis was performed on the ensemble of 400 structures resulting from the sampling of different mutual orientations of both TisB strands with respect to each other. It turned out that all structures ended up in a bound state, indicating that TisB is in fact capable of forming an antiparallel assembly inside the hydrophobic bilayer. The two most populated clusters contained 55 and 187 structures, respectively, and their central structures were selected for further refinement by means of all-atom MD simulations extended to 1 µs. The resulting geometries are shown in Fig. 2. Both clusters exhibited a similar structural pattern that was stable over the entire unrestrained MD simulation of 1 µs. RMSD of backbone atomic positions and SASA from the MD trajectories are shown in Tab. S1 and Fig. S1. The individual TisB strands – with an exception of several amino acid residues at both termini – retained an α-helical conformation throughout the entire simulation. The TisB monomers interact via the helix face carrying the strip of the charged and polar residues, while the hydrophobic surfaces point into the lipid environment. Rather than being perfectly upright in the membrane, the TisB strands 10


Page 10 of 30

Page 11 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2: Two most significant structures of the antiparallel TisB dimer assembly as obtained from DAFT, clusters 1 and 2, refined by means of extended all-atom MD simulations. Left – final snapshots after simulations of 1 µs. Right – averaged structures from these simulations, front and side views. Peptide backbones – gray ribbons, charged and polar side chains – colored sticks (Asp – red, Lys – blue, Gln – cyan), lipid molecules – yellow, water molecules close to peptide termini – colored by atom; more distant water molecules are omitted for clarity. of the dimer assembly are tilted at ca. 35° from the bilayer normal, see also Tab. S2. The termini of both dimer assemblies, which are located in the region of lipid head groups, are well solvated by water molecules that penetrate partly into this region. A slight difference between the two structures is that the TisB strands are in a closer contact in cluster 1, and the assembly appears nearly perfectly centro-symmetric. The dimer assembly in cluster 2 is less tilted from the membrane normal, at 33° as compared to 37° in cluster 1. Also, the TisB dimer in cluster 2 appears somewhat more loose than in cluster 1, as illustrated by its averaged SASA of 59.7 nm2 being larger than 52.5 nm2 for cluster 1. To characterize the interaction patterns between the TisB helices in detail, the distances between the charged and Gln side chains were measured. For the salt bridges, the distance between the Lys nitrogen and the Asp carboxyl carbon was considered; tentative hydrogen 11



bonds were described by the distance between the carbons of Gln amide groups, and between the Gln amido carbon and the Lys nitrogen. By this choice, the length of a tight salt bridge is 0.4 nm, as is that of a hydrogen bond between a Lys and a Gln, while a close interaction Q19–Q19 corresponds to a distance of 0.5–0.6 nm. The distances measured along all-atom MD simulations of TisB dimer in cluster 1 are presented in Fig. 3 (left). Similar data for cluster 2 are presented in Fig. S2 (left).

Figure 3: Left – lengths of salt bridges and hydrogen-bonded contacts in the TisB dimer, cluster 1, monitored along the all-atom MD simulation of 1 µs. Right – penetration of peptide and water into the lipid bilayer monitored by the one-dimensional densities along the bilayer normal, obtained for the respective groups of atoms. Data averaged along the all-atom MD simulations of 1 µs. Vertical distance of zero corresponds to the bilayer center. Let us assess the emergence of the tentative charge-zipper interaction pattern. As a first point, the salt bridges D5–K26 and K26–D5 only exist in the initial structures, which originated from the DAFT procedure. After several hundred ns of all-atom MD simulation, these salt bridges break and merely one of them appears again briefly (D5–K26 in cluster 2). On the other hand, the more centrally located salt bridges K12–D22 and D22–K12 show close contacts in both clusters, with distances that fluctuate in the range 0.4–0.7 nm. This indicates that there is in fact a salt-bridge interaction of these residues, however it is not a rigid one – rather, these residues are quite flexible. The remaining participant in the interaction of side chains is the centrally located Q19. The previously postulated Q19–Q19 hydrogen bond is not observed, while Q19 is engaged in hydrogen bonding with K12 instead; these observations were made in the simulations of 12


Page 12 of 30

Page 13 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


both structures. Therefore, the positively charged K12 possesses two interaction partners, i.e., the negatively charged D22 and the neutral Q19, hence we may think of it as a ternary interaction between K12(1), D22(2) and Q19(2). Also, the observed fluctuations of interstrand distances indicate a significant flexibility of the participating amino acid side chains. Therefore, this first model does not support a rigid charge-zipper pattern for the structure of an isolated TisB dimer. Notably, the side chains of both Q19 point away from each other, and the center of the interface between the TisB strands is thus free from any polar residues. As noted above, the TisB assembly is well hydrated at the termini. Thus, it is of interest to analyze how deeply the solvent penetrates into the lipid bilayer, and whether a certain amount of water even reaches the hydrophobic region. To this end, one-dimensional distributions of atoms along the bilayer normal were evaluated separately for the hydrophobic tails of lipids, the hydrophilic head groups of lipids, the TisB peptide component, and the solvent water. The resulting densities are presented in Fig. 3 (right), and similar data for the TisB dimer assembly in cluster 2 are presented in Fig. S2 (right). The antiparallel TisB dimer appears to be inserted symmetrically in the lipid bilayer, as expected, as indicated by its density centered around zero, which is the geometric center of the bilayer. The density of water extends into the hydrophobic region of lipid tails to some extent, and it vanishes entirely in the region of about −0.5 . . . 0.5 nm, in both structures. Recall that the side chains of Q19 point up and down, away from the the bilayer center, as if trying to reach the lipid head groups, so that the central segment of the TisB dimer does not contain any polar amino acid side chains. Thus, the central part of the TisB dimer of approximately 1 nm length, when embedded into the lipid bilayer, is completely hydrophobic and free of water molecules.

Experimental results To characterize the oligomeric assembly of TisB experimentally, we performed complementary circular dichroism (CD) spectroscopy and polyacrylamide gel electrophoresis (SDS13



PAGE) analyses in detergent micelles. Peptides were synthesized and purified as previously described. 6

Figure 4: Experimental results obtained on TisB. Left – circular dichroism spectrum. Right – gel from SDS-PAGE, left lane – molecular weight markers, right lane – TisB sample.

The CD spectrum of TisB in membrane-mimicking sodium dodecyl sulfate (SDS) micelles is shown in Fig. 4 (left). The line shape is characteristic of an α-helix, with two negative bands centered around 222 and 208 nm respectively, and a positive band at 192 nm, when measured in 10mM SDS at 25 °C. The result of an SDS-PAGE analysis of TisB is shown in Fig. 4 (right). It indicates the ability of TisB to oligomerize in the presence of SDS. Since TisB has a molecular weight of 3.2 kDa, the distinct band centered around 12 kDa indicates the tendency of TisB to assemble as a tetramer. Thus, it was demonstrated that TisB folds into an α-helix, and that TisB assembles as a tetramer in a membrane mimicking environment.

TisB tetramer Next, the DAFT procedure was performed to explore any possible tetrameric assemblies of TisB. DAFT employed simulations that started from 400 different orientations of four separate TisB strands; they will be denoted as A, B, C, D here, and A and C were oriented with the N-termini at the top of the lipid bilayer, while B and D took the opposite orientation, with the N-termini at the bottom. 313 out of 400 DAFT simulations resulted in a formation of a tetrameric assembly, and the final structures from these simulations were processed with 14


Page 14 of 30

Page 15 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


a cluster analysis. Clusters 1 and 2 consisted of 53 and 66 structures, respectively, and exhibited the most compact tetramer assemblies. Their mean structures were taken as starting geometries for subsequent all-atom MD simulations, which were extended to 2 µs. Both of these tetrameric TisB assemblies were stable over the entire simulation time, and their averaged structures are presented in Fig. S3. The orientations of the backbones of TisB strands remained quite stable in the simulations, leading to meaningful averaged structures, whereas amino acid side chains were rather dynamic, largely blurring their averaged orientations; see Tab. S3 for backbone RMSD data. In both structures, there were two TisB sub-dimers, constituting an X-shaped, lefthanded dimer-of-dimers, see Fig. 5 (top), and the tetrameric assembly was fully embedded into the POPC bilayer, see Fig. 5 (bottom). Each of the individual TisB strands assumed a predominantly α-helical conformation in the stretch of residues 5–26, see Fig. S4, and was tilted by 35–40° from the normal direction of lipid bilayer, see Tab. S4. In each of both subdimers, two TisB strands in antiparallel orientation interacted closely via salt bridges in a charge-zipped fashion. The two sub-dimers were also cross-linked by some weaker salt-bridge and hydrogen-bond contacts in the center of the assembly, including all four Q19 residues and the nearby charged residues. This leads to the appearance of a polar ‘tunnel’ extending from the aqueous phase into the peptide assembly, and across the entire thickness of the lipid bilayer. The structures of clusters 1 and 2 are strikingly similar, see also Tab. S3 and Fig. S5 for the corresponding backbone RMSD values. The only clear difference is that cluster 1 is composed of dimers A–D and C–B, while cluster 2 contained dimers A–B and C–D. This is clearly the consequence of the symmetry of the system: The strands B and D are oriented parallel in the bilayer, as are A and C – thus, the two sets of sub-dimers obtained from DAFT may be in principle identical. To analyze the structures in more detail, the contacts between the significant residues were analyzed in the same way as for the TisB dimer above. The distances between the charged and glutamine side chains were measured along the all-atom

15



Figure 5: The arrangement of TisB strands in the tetrameric assembly embedded in a POPC bilayer. Top – averaged structure from an all-atom MD simulation (TisB tetramer, cluster 2) viewed from the side (direction of bilayer plane) and from the top (direction of bilayer normal); peptide backbones – colored ribbons (strand A – blue, B – red, C – cyan, D – orange) with N- and C-termini labeled, charged and polar side chains – gray sticks and colored balls (Asp – red, Lys – blue, Gln – green). Bottom – a representative snapshot from that simulation showing the interaction with the lipid bilayer; peptide backbones – yellow ribbons, POPC head groups – colored by atoms. MD trajectories of both clusters 1 and 2. First, the tentative salt bridges and hydrogen bonds within the constituent charge-zipped sub-dimers were analyzed, and the results are presented in Fig. 6 and Tab. 1. The rather centrally located salt bridges K12–D22 and D22–K12 exhibit average distances of at most 0.43 nm with small standard deviations. This means that these contacts are present most of the time during the simulations. On the other hand, the larger average distances of 0.56–0.91 nm for D5–K26 and K26–D5 illustrate that these terminally located contacts only exist in certain intervals of the MD trajectories, while they are loosened during the remainder of the simulation time. This is apparent in the graphical representation of these distances: there are large and frequent fluctuations, indicating the flexible character 16


Page 16 of 30

Page 17 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6: Lengths of salt-bridged and hydrogen-bonded contacts in the charge-zipped subdimers of the TisB tetramer. Distances between the reference atoms (Nζ in the side chain of K12, Cδ in Q19, and Cγ in D22) measured along all-atom MD simulations of structures from clusters 1 and 2. Table 1: Lengths of salt bridges and hydrogen bonds in and between the subdimers of TisB within the tetramer. Average values ± std. deviations in nm over all-atom MD trajectories of 2 µs. contact D5–K26 K12–D22 (K12–Q19) Q19–Q19 (Q19–K12) D22–K12 K26–D5

tetramer charge-zipped sub-dimer A–D C–B 0.75 ± 0.41 0.38 ± 0.12 0.46 ± 0.12 0.36 ± 0.04 0.72 ± 0.22 0.92 ± 0.09 0.72 ± 0.23 0.54 ± 0.10 0.64 ± 0.17 0.54 ± 0.19 0.43 ± 0.11 0.43 ± 0.12 0.68 ± 0.39 0.89 ± 0.42

cluster 1 additional contacts A–B C–D 0.78 ± 0.32 1.45 ± 0.34 0.62 ± 0.16 1.30 ± 0.41 0.80 ± 0.33

0.80 ± 0.22 0.48 ± 0.11 1.45 ± 0.21 0.86 ± 0.26 1.02 ± 0.37

tetramer charge-zipped sub-dimer A–B C–D 0.73 ± 0.41 0.79 ± 0.40 0.41 ± 0.10 0.43 ± 0.13 0.64 ± 0.14 0.72 ± 0.17 0.79 ± 0.15 0.71 ± 0.19 0.46 ± 0.13 0.68 ± 0.16 0.38 ± 0.07 0.40 ± 0.12 0.91 ± 0.33 0.56 ± 0.27

cluster 2 additional contacts A–D C–B 1.09 ± 0.20 1.14 ± 0.27 0.98 ± 0.13 0.58 ± 0.12 0.43 ± 0.13

0.37 ± 0.06 1.24 ± 0.17 0.89 ± 0.21 0.66 ± 0.20 1.13 ± 0.21

of these salt bridges, which however appear repeatedly in the course of the simulations, and it seems that these contacts are never entirely lost. Apart from the charge-zipped sub-dimers, additional salt bridges D22–K12, and to a lesser extent K12–D22, are observed between the other antiparallel pairs of TisB helices: A–B & C–D in cluster 1, and A–D & C–B in cluster 2. These seem to contribute largely to the stabilization of the tetramer, which is, however, surely also driven by hydrogen bonding around the centrally located Q19 residues. To analyze the latter contribution, the distances of amino acid side chains in this region were measured, and the resulting values are presented in Tab. 2.

17



Table 2: Lengths of possible hydrogen-bonded contacts in the vicinity of the central Q19 residues in the TisB tetramer. Average values, in nm, obtained over the all-atom MD simulations of 2 µs. Reference atoms for measurement were Nζ in the side chain of K12, Cδ in Q19, and Cγ in D22. Shown are data for tentative contacts with averaged distances below 1.0 nm. contact K12–Q19 Q19–D22 Q19–Q19 Q19–K12 D22–Q19

pair of TisB monomers in cluster 1 A–B A–C A–D B–C B–D C–D 0.72 0.54 0.48 0.82 0.62 0.89 0.72 0.54 0.95 0.85 0.64 0.92 0.91 0.86 0.73 0.61 0.53

pair of TisB monomers in cluster 2 A–B A–C A–D B–C B–D C–D 0.64 0.66 0.72 0.63 0.79 0.51 0.98 0.89 0.80 0.71 0.46 0.58 0.68 0.93

While there are few strong, pertinent contacts observed (with distances of 0.4 nm, or 0.6 nm if Q19 participates), many averaged distances are around 0.7 nm. Although there do not seem to be distinctly pairwise and strong contacts between the centrally located amino acids, the involved Gln, Lys and Asp residues nevertheless seem to participate in a network of more variable and flexible contacts. These weaker effects add up to contribute to the association of the two sub-dimers of TisB, which is demonstrated by the tetramer assembly being stable in microsecond all-atom MD simulations.

Figure 7: Penetration of the TisB tetramer assembly and of water into the lipid bilayer according to their one-dimensional densities along the bilayer normal. Data averaged along all-atom MD simulations of 2 µs of TisB tetramer, clusters 1 and 2 from the DAFT ensemble. Vertical distance of zero corresponds to the geometric center of the bilayer. What needs to be clarified also is whether and how the solvent water can penetrate into the TisB tetramer or into the interior of the lipid bilayer surrounding the TisB bundle. To this end, one-dimensional densities for the components of the system were generated in the same manner as performed for the TisB dimers above. The resulting densities are presented 18


Page 18 of 30

Page 19 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


in Fig. 7, and they are in fact very similar to those observed for the isolated TisB dimer in Fig. 3 (right). The only noticeable difference is that while the density of water decreases when moving towards the center of the bilayer, it never really reaches zero. This means that there are always water molecules penetrating into the center of the TisB tetramer assembly, although in no large number as indicated by the non-zero but small values of density. These water molecules engage in interactions among the charged and Gln side chains along the polar ‘tunnel’ at the center of the TisB assembly. The water density decreases from both sides towards the zero of the vertical coordinate, hinting at an hourglass-shaped body of water molecules. To focus on the hydration of this ‘tunnel’, the water molecules present in it were counted, see Fig. 8. Even in the least hydrated part of the TisB assembly, which is in the center of the lipid bilayer, there are never fewer than 0.4 molecules per interval of 0.1 nm. This means that there is at least one water molecule per 0.3 nm present. Taking into account the oxygen–oxygen distance of 0.3 nm between two hydrogen-bonded water molecules, this amount of water is sufficient to form a continuous, hydrogen-bonded wire of water molecules. Note that this arrangement of water molecules is highly dynamic as illustrated by the large standard deviations in Fig. 8. Still, such a water wire may support proton transfer by means of Grotthuss mechanism and/or hydroxyl transfer mechanism.

Discussion and conclusion In our first step, the refinement of DAFT-based structures of an isolated antiparallel TisB dimer led to stable assemblies spanning the entire width of a POPC bilayer, tilted slightly away from the bilayer normal. The two most frequently occurring structures exhibited the same interaction pattern in these purely dimeric forms but there was no indication of a fully pronounced charge zipper. While two centrally located salt bridges K12–D22 were observed, any interactions closer to the termini were rather loose, and the entire structure appeared quite flexible in general. There was no contact between the two Q19 residues, instead they

19



Figure 8: Number of water molecules present within the TisB tetramer assembly, counted in bins of width of 0.1 nm along the bilayer normal. (A value of one means that a segment of the tetramer assembly of 0.1nm width along the membrane normal contains one water molecule on average.) Data averaged along all-atom MD simulations of 2 µs of TisB tetramer, clusters 1 and 2. Vertical distance of zero corresponds to the geometric center of the bilayer. Error bars denote standard deviations. engaged in hydrogen bonding with K12 of the opposite helix. The center of this assembly was entirely hydrophobic, free of any polar residues and water. Obviously, this dimeric structure would not support the conductance of ions or the breakdown of a pH gradient. The DAFT procedure coupled with all-atom MD simulation using a state-of-the-art force field seems to be a suitable tool to refine the structure of an oligomeric membrane-active peptide with a given order, and can reveal the helix orientation in a lipid bilayer. However, even the acceleration of sampling by means of DAFT is still insufficient to predict the actual oligomeric state of a complex system such as a peptide in a lipid bilayer, and the secondary structure of the individual protomers. These questions have to be tackled by means of experimental approaches. Here, TisB was confirmed to assume α-helical conformation with CD spectroscopy in a membrane mimicking environment, and SDS-PAGE analysis revealed that it actually forms stable tetramer assemblies. Note that the previous report of a dimeric state was based on a Hill coefficient of two, 6 which in fact also complies with a higher multiple of two, such as a tetramer. In the same report, it was also shown that TisB remains helical in lipid vesicles, and that the helix is oriented upright in the membrane. Put together, all of 20


Page 20 of 30

Page 21 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


these data suggest a tetramer assembly of TisB, i.e., a dimer composed of two antiparallel TisB dimers that could be themselves stabilized by charge-zipper interactions and hydrogen bonding. The structure and orientation of tetramer assemblies of TisB were investigated here with an approach combining DAFT and unrestrained all-atom MD simulations. The DAFT started from four separate monomers, and was thus not biased towards the formation of any specific oligomeric assembly. Clustering analysis revealed that two of the dominant clusters from DAFT in fact represent one and the same structure, up to an exchange of the individual TisB strands, which is possible due to symmetry. This tetrameric structure of TisB exhibits an interesting and robust interaction pattern, which was further refined and described on the basis of all-atom simulations. The antiparallel tetrameric bundle (up-down-up-down) is composed of two tightly bound sub-dimers of TisB, which are assembled into a left-handed X-shaped dimer-of-dimers. Each of the dimers features a full charge-zipped pattern involving four Lys–Asp salt bridges, 7 and all monomers are in close contact with one another in the central region that includes Gln19. There is a rich network of hydrogen bonds, and a few additional, cross-linking salt bridges are furthermore observed between the pairs of dimers, see Fig. 9 (top and left). Also, a small amount of solvent water is found to penetrate deeply into the center of the tetramer assembly, see Fig. 9 (right). Unlike the isolated dimer discussed above, this tetrameric structure of TisB could conceivably support the conductance of ions or the breakdown of a pH gradient, which was demonstrated experimentally 5 and constitutes the molecular mechanism of biofilm induction. 16

Acknowledgement The authors acknowledge Andrea Eisele, Kerstin Scheubeck, Bianca Posselt and Ronja Kammerichs for assistance in peptide synthesis, purification, CD and SDS-PAGE. This work was supported by the German Research Foundation (DFG) through grants GRK 2039, UL 172/721



Figure 9: Top – contacts between the amino acid side chains observed in the simulations of the TisB tetramer, based on distances from the all-atom MD simulation of cluster 1; purple – salt bridges, green – hydrogen bonds, solid line – average distance < 0.5 nm (or 0.7 nm wherever Q19 participates), dashed line – average distance < 0.8 nm (or 1.0 nm wherever Q19 participates). Left – important contacts in the structure of the TisB tetramer; colored balls – charged and polar side chains (red – Asp, blue – Lys, green – Gln). Right – water wire in a representative structure from the all-atom MD simulation of cluster 2. 1 and KU 3677/2. The computations were performed partly on the JUSTUS HPC facility, supported by the state of Baden-W¨ urttemberg through bwHPC and by the DFG through grant INST 40/467-1 FUGG.

Supporting Information Available Supporting methods – details of the DAFT procedure and of all-atom MD simulations. Additional data on TisB dimer – RMSD of backbone atom positions, SASA, tilt angles, lengths of interstrand contacts and penetration into lipid bilayer of TisB dimer – cluster 2.

22


Page 22 of 30

Page 23 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Additional data on TisB tetramer – views of averaged structures of TisB tetramer cluster 1 and 2, helicity of TisB strands, RMSD of backbone atom positions, SASA, tilt angles. References 53–60 appear in the Supporting Information as Refs. S1–S8.

This material is

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

References (1) Dörr, T.; Vulić, M.; Lewis, K. Ciprofloxacin causes persister formation by inducing the TisB toxin in Escherichia coli. PLoS Biol. 2010, 8, e1000317. (2) Wagner, E. G. H.; Unoson, C. The toxin-antitoxin system tisB-istR1. Expression, regulation and biological role in persister phenotypes. RNA Biol. 2012, 9, 1513–1519. (3) Unoson, C.; Wagner, E. G. H. A small SOS-induced toxin is targeted against the inner membrane in Escherichia coli. Mol. Microbiol. 2008, 70, 258–270. (4) Yamaguchi, Y.; Park, J.-H.; Inouye, M. Toxin-antitoxin systems in bacteria and archaea. Annu. Rev. Genet. 2011, 45, 61–79. (5) Gurnev, P. A.; Ortenberg, R.; Dörr, T.; Lewis, K.; Bezrukov, S. M. Persister-promoting bacterial toxin TisB produces anion-selective pores in planar lipid bilayers. FEBS Lett. 2012, 586, 2529–2534. (6) Steinbrecher, T.; Prock, S.; Reichert, J.; Wadhwani, P.; Zimpfer, B.; B¨ urck, J.; Berditsch, M.; Elstner, M.; Ulrich, A. S. Peptide-lipid interactions of the stress-response peptide TisB that induces bacterial persistence. Biophys. J. 2012, 103, 1460–1469. (7) Walther, T. H.; Ulrich, A. S. Transmembrane helix assembly and the role of salt bridges. Curr. Opin. Struct. Biol. 2014, 27, 63–68. (8) Walther, T.; Gottselig, C.; Grage, S.; Wolf, M.; Vargiu, A.; Klein, M.; Vollmer, S.; Prock, S.; Hartmann, M.; Afonin, S. et al. Folding and self-assembly of the TatA translocation pore based on a charge zipper mechanism. Cell 2013, 152, 316–326. 23



(9) Zhou, S.; Pettersson, P.; Huang, J.; Sjöholm, J.; Sjöstrand, D.; Pomès, R.; Högbom, M.; ¨ Brzezinski, P.; Mäler, L.; Adelroth, P. Solution NMR structure of yeast Rcf1, a protein involved in respiratory supercomplex formation. Proc. Natl. Acad. Sci. USA 2018, 115, 3048–3053. (10) Kandt, C.; Ash, W. L.; Tieleman, D. P. Setting up and running molecular dynamics simulations of membrane proteins. Methods 2007, 41, 475–488. (11) Lindahl, E.; Sansom, M. S. P. Membrane proteins: molecular dynamics simulations. Curr. Opin. Struct. Biol. 2008, 18, 425–431. (12) Rzepiela, A. J.; Sengupta, D.; Goga, N.; Marrink, S.-J. Membrane poration by antimicrobial peptides combining atomistic and coarse-grained descriptions. Faraday Discuss. 2010, 144, 431–443. (13) Wang, Y.; Zhao, T.; Wei, D.; Strandberg, E.; Ulrich, A. S.; Ulmschneider, J. P. How reliable are molecular dynamics simulations of membrane active antimicrobial peptides? Biochim. Biophys. Acta Biomem. 2014, 1838, 2280–2288. (14) Strandberg, E.; Ulrich, A. S. In Modern magnetic resonance; Webb, G. A., Ed.; Springer: Cham, 2017; pp 1985–1996. (15) Strandberg, E.; Ulrich, A. S. In Modern magnetic resonance; Webb, G. A., Ed.; Springer: Cham, 2017; pp 651–667. (16) Berditsch, M.; Reichert, J.; Sanyal, P.; Wadhwani, P.; Zimpfer, B.; Grage, S. L.; Xu, X.; Richter, K.; Wagner, M.; Schleicher, A. et al. Bacterial stress-response peptide TisB: charge-zipper structure, molecular mechanism of biofilm-induction, and application in biological fuel cells. in preparation. (17) Reißer, S.; Strandberg, E.; Steinbrecher, T.; Elstner, M.; Ulrich, A. S. Best of two worlds? How MD simulations of amphiphilic helical peptides in membranes can com24


Page 24 of 30

Page 25 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


plement data from oriented solid-state NMR. J. Chem. Theory Comput. 2018, 14, 6002–6014. (18) Feller, S. In Lipid bilayers: Structure and interactions; Katsaras, J., Gutberlet, T., Eds.; Springer: Berlin, Heidelberg, 2001; pp 89–107. (19) Berendsen, H. J. C.; Tieleman, D. P. In Encyclopedia of computational chemistry; von Ragué Schleyer, P., Ed.; Wiley: New York, 1998; Vol. 3; pp 1639–1650. (20) Lyubartsev, A. P.; Rabinovich, A. L. Force field development for lipid membrane simulations. Biochim. Biophys. Acta Biomembranes 2016, 1858, 2483–2497. (21) Chipot, C.; Pohorille, A. Structure and dynamics of small peptides at aqueous interfaces: A multi-nanosecond molecular dynamics study. J. Mol. Struct. Theochem 1997, 398-399, 529–535. (22) Chipot, C.; Pohorille, A. Folding and translocation of the undecamer of poly-L-leucine across the water-hexane interface. A molecular dynamics study. J. Am. Chem. Soc. 1998, 120, 11912–11924. (23) Tieleman, D. P.; Berendsen, H. J. C.; Sansom, M. S. P. Surface binding of alamethicin stabilizes its helical structure: Molecular dynamics simulations. Biophys. J. 1999, 76, 3186–3191. (24) Bond, P. J.; Holyoake, J.; Ivetac, A.; Khalid, S.; Sansom, M. S. P. Coarse-grained molecular dynamics simulations of membrane proteins and peptides. J. Struct. Biol. 2007, 157, 593–605. (25) Marrink, S. J.; Risselada, H. J.; Yefimov, S.; Tieleman, D. P.; de Vries, A. H. The MARTINI force field: Coarse grained model for biomolecular simulations. J. Phys. Chem. B 2007, 111, 7812–7824.

25



(26) Monticelli, L.; Kandasamy, S. K.; Periole, X.; Larson, R. G.; Tieleman, D. P.; Marrink, S.-J. The MARTINI coarse-grained force field: Extension to proteins. J. Chem. Theory Comput. 2008, 4, 819–834. (27) Wassenaar, T. A.; Pluhackova, K.; Moussatova, A.; Sengupta, D.; Marrink, S. J.; Tieleman, D. P.; Böckmann, R. A. High-throughput simulations of dimer and trimer assembly of membrane proteins. The DAFT Approach. J. Chem. Theory Comput. 2015, 11, 2278–2291. (28) Parton, D. L.; Akhmatskaya, E. V.; Sansom, M. S. P. Multiscale simulations of the antimicrobial peptide maculatin 1.1: Water permeation through disordered aggregates. J. Phys. Chem. B 2012, 116, 8485–8493. (29) Horn, J. N.; Romo, T. D.; Grossfield, A. Simulating the mechanism of antimicrobial lipopeptides with all-atom molecular dynamics. Biochemistry 2013, 52, 5604–5610. (30) Lin, D.; Grossfield, A. Thermodynamics of antimicrobial lipopeptide binding to membranes: Origins of affinity and selectivity. Biophys. J. 2014, 107, 1862–1872. (31) Friess, M. D.; Pluhackova, K.; Böckmann, R. A. Structural model of the mIgM B-cell receptor transmembrane domain from self-association molecular dynamics simulations. Front. Immunol. 2018, 9, 2947. (32) Gurtovenko, A. A.; Anwar, J.; Vattulainen, I. Defect-mediated trafficking across cell membranes: Insights from in silico modeling. Chem. Rev. 2010, 110, 6077–6103. (33) Hub, J. S.; de Groot, B. L.; Grubm¨ uller, H.; Groenhof, G. Quantifying artifacts in Ewald simulations of inhomogeneous systems with a net charge. J. Chem. Theory Comput. 2014, 10, 381–390. (34) Case, D. A.; Betz, R. M.; Cerutti, D. S.; Cheatham III, T. E.; Darden, T. A.;

26


Page 26 of 30

Page 27 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Duke, R. E.; Giese, T. J.; Gohlke, H.; Goetz, A. W.; Homeyer, N. et al. AmberTools 2016. University of California, San Francisco, CA, 2016. (35) de Jong, D. H.; Singh, G.; Bennett, W. F. D.; Arnarez, C.; Wassenaar, T. A.; Schäfer, L. V.; Periole, X.; Tieleman, D. P.; Marrink, S. J. Improved parameters for the Martini coarse-grained protein force field. J. Chem. Theory Comput. 2013, 9, 687–697. (36) Wassenaar, T. A.; Pluhackova, K.; Böckmann, R. A.; Marrink, S. J.; Tieleman, D. P. Going backward: A flexible geometric approach to reverse transformation from coarse grained to atomistic models. J. Chem. Theory Comput. 2014, 10, 676–690. (37) Wolf, M. G.; Hoefling, M.; Aponte-Santamar´ıa, C.; Grubm¨ uller, H.; Groenhof, G. g membed: Efficient insertion of a membrane protein into an equilibrated lipid bilayer with minimal perturbation. J. Comput. Chem. 2010, 31, 2169–2174. (38) Maier, J. A.; Martinez, C.; Kasavajhala, K.; Wickstrom, L.; Hauser, K. E.; Simmerling, C. ff14SB: Improving the accuracy of protein side chain and backbone parameters from ff99SB. J. Chem. Theory Comput. 2015, 11, 3696–3713. (39) Jämbeck, J. P. M.; Lyubartsev, A. P. Derivation and systematic validation of a refined all-atom force field for phosphatidylcholine lipids. J. Phys. Chem. B 2012, 116, 3164– 3179. (40) Jämbeck, J. P. M.; Lyubartsev, A. P. An extension and further validation of an allatomistic force field for biological membranes. J. Chem. Theory Comput. 2012, 8, 2938–2948. (41) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 1983, 79, 926–935.

27



(42) Joung, I. S.; Cheatham, T. E. Determination of alkali and halide monovalent ion parameters for use in explicitly solvated biomolecular simulations. J. Phys. Chem. B 2008, 112, 9020–9041. (43) van der Spoel, D.; Lindahl, E.; Hess, B.; Groenhof, G.; Mark, A. E.; Berendsen, H. J. C. GROMACS: Fast, flexible, and free. J. Comput. Chem. 2005, 26, 1701–1718. (44) Hess, B.; Kutzner, C.; van der Spoel, D.; Lindahl, E. GROMACS 4: Algorithms for highly efficient, load-balanced, and scalable molecular simulation. J. Chem. Theory Comput. 2008, 4, 435–447. (45) Abraham, M. J.; Murtola, T.; Schulz, R.; Páll, S.; Smith, J. C.; Hess, B.; Lindahl, E. GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 2015, 1-2, 19–25. (46) Tribello, G. A.; Bonomi, M.; Branduardi, D.; Camilloni, C.; Bussi, G. PLUMED 2: New feathers for an old bird. Comput. Phys. Commun. 2014, 185, 604–613. (47) Humphrey, W.; Dalke, A.; Schulten, K. VMD – Visual Molecular Dynamics. J. Mol. Graphics 1996, 14, 33–38. (48) xmGrace 5.1.24. http://plasma-gate.weizmann.ac.il/Grace (accessed December 16, 2018). (49) Eisenhaber, F.; Lijnzaad, P.; Argos, P.; Sander, C.; Scharf, M. The double cubic lattice method: Efficient approaches to numerical integration of surface area and volume and to dot surface contouring of molecular assemblies. J. Comput. Chem. 1995, 16, 273–284. (50) M¨ uhlhäuser, P.; Wadhwani, P.; Strandberg, E.; B¨ urck, J.; Ulrich, A. S. Structure analysis of the membrane-bound dermcidin-derived peptide SSL-25 from human sweat. Biochim. Biophys. Acta Biomembr. 2017, 1859, 2308–2318.

28


Page 28 of 30

Page 29 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(51) Windisch, D.; Hoffmann, S.; Afonin, S.; Vollmer, S.; Benamira, S.; Langer, B.; B¨ urck, J.; Muhle-Goll, C.; Ulrich, A. S. Structural role of the conserved cysteines in the dimerization of the viral transmembrane oncoprotein E5. Biophys. J. 2010, 99, 1764–1772. (52) Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227, 680–685. (53) Bussi, G.; Donadio, D.; Parrinello, M. Canonical sampling through velocity rescaling. J. Chem. Phys. 2007, 126, 014101. (54) Berendsen, H. J. C.; Postma, J. P. M.; van Gunsteren, W. F.; DiNola, A.; Haak, J. R. Molecular dynamics with coupling to an external bath. J. Chem. Phys. 1984, 81, 3684– 3690. (55) Darden, T.; York, D.; Pedersen, L. Particle mesh Ewald – an n · log(n) method for Ewald sums in large systems. J. Chem. Phys. 1993, 98, 10089–10092. (56) Nosé, S. A unified formulation of the constant temperature molecular-dynamics methods. J. Chem. Phys. 1984, 81, 511–519. (57) Hoover, W. G. Canonical dynamics: Equilibrium phase-space distributions. Phys. Rev. A 1985, 31, 1695–1697. (58) Nosé, S.; Klein, M. L. Constant pressure molecular dynamics for molecular systems. Mol. Phys. 1983, 50, 1055–1076. (59) Hess, B.; Bekker, H.; Berendsen, H. J. C.; Fraaije, J. G. E. M. LINCS: A linear constraint solver for molecular simulations. J. Comput. Chem. 1997, 18, 1463–1472. (60) Páll, S.; Hess, B. A flexible algorithm for calculating pair interactions on SIMD architectures. Comput. Phys. Commun. 2013, 184, 2641–2650.

29



Graphical TOC Entry

30


Page 30 of 30

Tetrameric Charge-Zipper Assembly of the TisB Peptide in

Recommend Documents