Preferred Binding Mechanism of Osh4's Amphipathic Lipid-Packing

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B: Biomaterials and Membranes

Preferred Binding Mechanism of Osh4’s ALPS Motif, Insights From Molecular Dynamics Viviana Monje-Galvan, and Jeffery B. Klauda J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b07067 • Publication Date (Web): 03 Oct 2018 Downloaded from http://pubs.acs.org on October 6, 2018

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The Journal of Physical Chemistry

Preferred Binding Mechanism of Osh4’s ALPS Motif, Insights From Molecular Dynamics Viviana Monje-Galvan1 and Jeffery B. Klauda1,2* 1

Department of Chemical and Biomolecular Engineering and 2Biophysics Program, University of Maryland, College Park, MD 20742, USA *To whom correspondence should be addressed: [email protected]

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Abstract Amphipathic helices are key domains of peripheral membrane proteins, targeting specific membranes to enable proper protein function as well as changing the local topology and lipid dynamics of the membranes they bind. Here we use extended all-atom molecular dynamics to study, in detail, the binding mechanism and conformation of the N-terminus of the lipid transport protein Osh4 in yeast, i.e., the amphipathic lipid packing sensor (ALPS) motif. We identified two binding conformations: (i) a vertical one with the N-terminus of the peptide embedded into the hydrophobic core, and (ii) a horizontal, and energetically favored, conformation in which the hydrophobic side chains of ALPS are fully embedded into the membrane hydrophobic core. From extensive analysis on 21 trajectories of 2µs each, we describe peptide binding in terms of the structural changes that both the peptide and the membrane undergo upon binding as well as energetics of this interaction. The membrane models in this study include a simple binary lipid mixture, with a neutral and a charged lipid (DOPC/DOPS), and complex mixtures with lipid compositions characteristic of two organelles in yeast (each with more than 6 lipid types and an accurate sterol content). Our conclusions are in agreement with available literature, showing the ALPS peptide is more likely to bind membrane surfaces with packing defects and higher anionic character. In addition, we show that there is an interplay between ALPS binding an existing packing defect and creating or enhancing one as the peptide binds to the membrane, which was previously suggested in the literature.

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Introduction Proteins are essential for all biological processes; they perform a variety of roles in the cell, from catalyzing reactions to transporting cargo or serving as mechanical support.1 Peripheral membrane proteins (PMPs) have received renewed attention in recent years as they are involved in cell signaling processes and cargo transport; a thorough review of the current state of research in this matter was recently published by Bassereau et al.2 Their distribution and activity on the membrane surface, a bilayer, is highly influenced by membrane lipids and different structural domains allow them to interact with specific membranes to achieve their function.3 Common membrane binding domains of PMPs are the PH (pleckstrin homology), CERT (ceramide transport protein), F-BAR (F-Bin-Amphiphysin-Rvs), and ALPS (amphipathic lipid packing sensors); some of which target specific lipid types such as phosphoinositides (PIs), sterols, and ceramides.4-6 Lipid-transport proteins (LTPs) are a specific subcategory of PMPs that provide alternate means to vesicular transport.6-8 LTPs may target a bilayer using curvature-sensing domains like amphipathic helices (AH) due to their distinct hydrophobic/non-polar and polar faces.5-6, 9 Amphipathic helices (AH) have received increased attention in the past 15 years due to their ability to bind curved membranes or induce curvature; sometimes they even constitute the main domain that enables membrane binding.6, 10 These smaller domains are at the core of studies on drug delivery

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and cell toxicity due to element insertion into the bilayer.12 Their interaction

with biological bilayers has been linked to membrane stress response, cell signaling, and lipid trafficking among others.8 Examples of helical peptide-membrane studies in the recent years include curvature sensors like ArfGAP1

9, 13-14

, CRAC peptides

peptide structure and bound conformation (MP-X study)

16

15

, model α-helices to study

, and hairpin virus peptides to study 3

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membrane fusion processes.17 However, the question still remains as to how they bind to a membrane and if they bind to a curved leaflet, induce curvature, or if there is a feed-back-loop between both.18-19 A suggested curvature-sensing mechanism is the folding of the AH upon contact with curved membranes through insertion of its large hydrophobic residues into the membrane core in places of loose lipid packing.9,

20

Antonny provides a thorough review on

curvature sensing domains and the proposed binding mechanisms of several ones.6 In recent years, the extent to which membrane lipid composition influences protein binding has drawn more attention, particularly the role of lipid rafts and sterols. 18, 20-22 ALPS motifs are a subcategory of AHs with characteristic abundance of Ser and large bulky hydrophobic residues as well as a low density of changed amino acids.9, 22 They are involved in cell stress response and membrane targeting for lipid trafficking, and bind preferably to membranes with large packing defects.22-24 Yet, their binding mechanism and regulation as well as the membrane response to peptide binding have not been studied in detail.21 In addition, the specificity of membrane targeting by ALPS peptides remains to be elucidated.20, 22 Here we use molecular dynamics (MD) simulations to examine the influence of membrane lipid composition on the binding mechanism of the ALPS motif of Osh4 in yeast Saccharomyces cerevisiae. Osh4 is one of seven oxysterol-binding protein homologues (Osh) family in yeast; this LTP is known to strongly bind membranes containing charged lipids, specifically PI(4)P, the major representative of PI lipids in the trans-Golgi network, and interchange it with sterol.25-31 The protein structure and mechanism of the Osh family in yeast has been used to model and understand that of the oxysterol-binding proteins (OSBP), their counterparts in humans, and other OSBP-related proteins (ORPs) in different organisms.32-33 These proteins perform relevant roles in cell signaling processes and are linked to cancer cell activity.34-35 The conserved domains


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across OSBPs and ORPs allow the study of selected members of these LTP families in order to gain general understanding of the mechanisms they have in common. The OSBPs and ORPs operate at membrane contact sites, therefore, it is important to understand the protein-membrane interactions and binding domains that enable proper membrane targeting and function. The ALPS motif of Osh4 is an AH formed by 27 amino acids that acts as a lid to protect sterols inside the binding pocket during transport (Fig. 1). This peptide is the N-terminus of the Osh4 protein and is one of its six membrane binding regions.29 The ALPS motif binds to the membrane through nonspecific interactions with anionic lipids and acts as a membrane curvature and/or lipid packing sensor.5, 9 Although ALPS is not necessarily an anchor for the full protein, experiments show reduced binding of Osh4 to small vesicles when this motif is not present.30 Whether ALPS binds to membranes due to their packing defects, or if it instead creates packing defects upon binding and thus facilitates lipid extraction for Osh4 remains to be determined. In this work we carried simulations to identified key residues that stabilize the binding of ALPS to model membranes and propose a stable bound conformation. Our results contribute to the understanding of factors that dominate protein-membrane interactions of curvature-sensing domains and AHs in general.



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Figure 1 - ALPS motif of Osh4: (A) helix wheel (generated with http://rzlab.ucr.edu/scripts/wheel); (B) location in the full protein; and (C) polar (green) and non-polar (white) residues of the peptide, residues in the main helix of the peptide are shown explicitly. The red dashed line indicates the main axis of the amphipathic helix; define from Cα-S8 to Cα-S18 to calculate the tilt angle of the peptide with respect to the membrane surface and the rotation of nonpolar face of the helix with respect to its main axis. The red arrow is the normal vector of the non-polar face.

Methods Systems buildup and simulation The binding mechanism of the ALPS motif of Osh4 was studied with a simple binary membrane model and with the symmetric endoplasmic reticulum (ER) and trans-Golgi network (TGN) membrane models we previously developed.36 The simple models consisted of a mixture of DOPC and DOPS lipids in a 3:2 ratio (60:40 mol %) in bilayers of 80 lipids per leaflet, to mimic the composition of anionic lipids in yeast membranes. The complex models had 150 lipids 6 ACS Paragon Plus Environment

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per leaflet and had different composition as listed in Table S1; details on membrane structure and mechanical properties for these were presented along with a model for the plasma membrane in 36

. Before incorporating the ALPS peptide, membrane models were built using the Membrane

Builder feature in CHARMM-GUI (www.charmm-gui.org).37-39 At least 30 molecules of water per lipid were used to ensure full hydration using the TIP3P model for water40 and neutralizing potassium ions. Each model was equilibrated with CHARMM41 using the typical six-step CHARMM-GUI protocol for 225 ps.38-39 All trajectories were run using the CHARMM36 lipid force field (C36),42 with the most updated parameters for PI lipids43 and sterols.44 Membraneonly trajectories were run in triplicates and initially equilibrated for at least 100ns on NAMD45 with a 2 fs time-step using the SHAKE algorithm to constraint hydrogen atoms.46 The temperature was kept constant at 303.15K to ensure a fluid phase membrane using the NoséHoover thermostat47 and Langevin dynamics.48-49 The cell box size varied semi-isotropically (X=Y but not Z) with constant pressure of 1bar using a Langevin piston.45, 50-51 van der Waals (VDW) and electrostatics were computed using a Lennard-Jones force-switching function over 10 to 12 Å.52 We used periodic boundary conditions (PBC) to evaluate long-range electrostatic interactions using Particle Mesh Ewald (PME).53 To build the ALPS-membrane systems, first the coordinates for the peptide were extracted from the full protein, PDBID: 1zhz,54 and briefly equilibrated in water using the Quick Solvator in CHARMMM-GUI. The peptide was inserted in the aqueous phase of the equilibrated membrane-only systems using CHARMM and at least 8 Å away from the bilayer; overlapping water molecules were deleted. A block of water was added using Packmol55 when needed to allow enough room for the peptide to freely rotate without forcing peptide-membrane interactions. Two initial peptide orientations were used to avoid biased binding events, either



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perpendicular or parallel to the bilayer normal. Since a previous study showed ALPS peptides bind to membranes with surface-packing defects,9 systems with and without surface tension were examined for all membrane models to further increase the lipid packing defects on the membrane surface. Doing so, exposed more of the membrane’s hydrophobic core56 and allowed us to examine this effect on the binding time scale, orientation, and mechanism. The NPT ensemble was used to run simulations without surface tension, and the NPγT ensemble was used with γ=20 dyn/cm of tension. 100-ns equilibration in NAMD was performed on these systems. To approach necessary µs timescales, the systems were converted into DESRES formant and extended to 2µs trajectories on the Anton Machine,57 for a total of 42µs of simulation among the 21 systems run, all listed in Table S2. C36 parameters for ergosterol (ERG) and PI lipids were added as custom parameters to the viparr library in Anton, including parameters for non-bonded interactions; i.e. NBFIX parameters from the C36 FF for sterols and PIs. Temperature and pressure controls for NPT dynamics were set using Anton’s multigrator 58 with a 2 fs time step, and the cut-off values for neighboring atoms was selected using Anton’s ark files (scripts to optimize the parameters for the integration algorithms of the simulation). Long-range electrostatics were computed using the Gaussian split Ewald algorithm on Anton

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with cutoffs

also set by Anton guesser scripts. The cut-off for non-bonded interactions was set to 10 Å and that for long-range electrostatics to 12 Å for the onpt, znpt, er, 20er, and 20tgn systems. The corresponding values for the onvt, and znvt systems were 9 Å and 9.6 Å for non-bonded and electrostatics, respectively; while the tgn system parameters were set by the Anton guessers to 12 Å and 14 Å. Replicate runs used the same cut-off values as the first run for each set for consistency. Anton software does not support a surface tension ensemble, therefore the systems equilibrated with NPγT dynamics on NAMD were extended using the NVT ensemble on Anton.


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System Analysis Binding events were examined qualitatively using Visual Molecular Dynamics (VMD)60 to determine the bound configuration of the peptide and get an estimate of the binding time scale. The interaction energies (∆Ebind) between the peptide and membrane were computed using CHARMM. These were block-averaged for each system, and then averaged across systems bound in the same final conformation (horizontal vs. vertical). In addition, the interaction energy between every residue in the peptide and the membrane was computed using CHARMM and the same cutoffs for non-bonded interactions as those used during the trajectory. The reported values are estimates of the enthalpic contribution to the free energy of binding; the entropic contributions are more complex and require extensive sampling that was out of the scope of this study. The values reported for the enthalpic contribution to peptide-membrane interaction are not an absolute measure of the free energy of this process, given by the Gibbs free energy for the NPT ensemble. As such, these estimates should be used with caution as the relative likelihood of observing the peptide bound in one or another conformation, and not as a measure to determine the driving force or reversibility of said interaction. In addition, without considering the entropic contribution to the overall free energy, one cannot fully conclude the spontaneity of a binding event; given the process is stochastic, we aim to answer the question of favorable binding examining 21 systems in this study. In addition to the qualitative analysis on VMD, this software package was used to track the rotation of the main helix (residues 8-18) of the peptide about itself. A normal vector was defined for the non-polar face of the peptide (see Fig 1C) and a time series generated to track the orientation of the non-polar side chains as the simulation progressed with respect to the normal vector of the membrane surface (blue arrow). Since the simulation was run using PBC and the 9 ACS Paragon Plus Environment


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peptide was free to move in water, it bound to the top and bottom leaflet of the bilayer indistinctly. The rotation angle is reported relative to the positive z-axis, the normal vector to the top leaflet. Fig. S1 shows a representation of the main helix rotation with respect to its main axis and the different values the rotation angle can take. The tilt angle of the main helix with respect to the membrane surface was computed for all systems upon stable binding using CHARMM. Electron density profiles (EDPs) were plotted to show the relative position of the lipid headgroups and the protein using the SIMtoEXP software package following the procedure of Kučerka et al.61 EDPs for the final location of the protein were computed and averaged over at least the last 500ns of each trajectory. These show the peptide’s location at the beginning and at the end of the simulation as well as the location of the full protein, N-terminus (residues 1 to 7), C-terminus (residues 19 to 27), and the main helix (residues 8 to 18) with respect to different regions in the membrane. Also to this purpose, the frequency of contact (FOC) of each residue with the membrane was computed during the last 700ns of simulation and blocked averaged every 60ns. CHARMM was used to count, respectively, the number of times a phosphorous atom (P) and the second carbon of glycerol (C2) in the lipids were within 5Å of the Cα atom of each residue in the peptide. Averaged values between all the simulations sharing a binding conformation (horizontal vs. vertical) are reported in the next section. Finally, the helical character of both the full peptide and the main helix throughout the trajectory was determined using CHARMM as the percent of amino acids with an α-helical secondary structure in the form of a time series. Data was collected every 0.2ns for the entire trajectory and then blocked every 10ns. Comparison was done between horizontal and vertical bound conformations as well as between simple and complex systems. Results were averaged across systems sharing a bound conformation unless stated otherwise.


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Results We used three membrane models to assess the binding mechanism and interaction of the ALPS motif with model membranes. The simplest model is a binary mixture of DOPC:DOPS (60:40) lipids to model an anionic bilayer, while complex bilayers with 6+ lipid species were used to mimic the ER and TGN membranes in yeast. Initial examination of the 21 trajectories using VMD showed most of the systems resulted in one of two bound conformations, discussed below, and remained in the bound conformation for at least 500 ns. In this section we describe the peptide-membrane interactions quantitatively and then discuss its preference for membrane character and binding orientation. To reiterate, this study used coordinates of the fully folded ALPS motif as truncated from the Osh4 protein (PDBID: 1zhz). The scope of this work did not examine the folding/unfolding transitions of the peptide at the membrane interface; however, two of our simulation trajectories did show partial unfolding of the C-terminus during peptide adsorption onto the binding leaflet – one of these systems is described in detail as an example. Binding conformations The ALPS motif bound the membrane models in two conformations independently of the initial orientation of the peptide above the bilayer. The peptide bound either vertically or slightly tilted with respect to the membrane surface (Fig. 2A), or horizontally with its non-polar face embedded into the bilayer at the phosphate region of the interacting leaflet (Fig. 2B). In the vertical conformation, the N-terminus interacts with the bilayer, the hydrophobic side chains are usually pointing towards the water (white face of the peptide in Fig.2), and the C-terminus is usually extended towards the solvent. In the horizontal conformation, the non-polar face of the peptide interacts with the hydrophobic core and the N-terminus is usually embedded into the membrane; 11 ACS Paragon Plus Environment


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Figure 2 – Peptide bound conformations for the (A) znpt2 system (vertical) and (B) znvt system (horizontal); (C)&(D) EDPs of the membrane, with each peak representing the average location of

the lipid headgroups of each leaflet, and sections of the peptide for each conformation. For (E) and (F): charged lipids shown in red and neutral lipids shown in cyan for the interacting leaflet, the opposite leaflet is shown in blue, and main interacting residues shown explicitly for the (E) 12 ACS Paragon Plus Environment

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vertical conformation showing Tyr4 interacting with the bilayer, and (F) horizontal conformation showing Lys15 in blue, and Trp10 and Phe13 in white. it is extended towards the water along with the C-terminus in 30% of the cases. The location of the peptide with respect to the lipid headgroups is also shown in the sample EDPs on Fig. 2C&D; final snapshots for every system are shown in Fig. S2-S5 along with the time series of the rotation angle of the main helix, which is discussed in more detail later on. The corresponding EDP for each system is shown in Figs. S6-S9. Two of the PC-PS systems, 15% of the simulations with simple membrane models, did not show any stable binding, rather an stochastic survey of the membrane surface with no permanent interaction with the bilayer (o-npt1 and z-npt1 as listed on Table S2). Since the peptide spent most of the time in the solvent or barely interacting with the membrane surface, it was free to rotate in any direction. Thus, the rotation angle of the main helix on its own axis shown in Fig. S2B and S3B did not stabilize and it is simply noise. Only two out of the eleven vertical orientations were bound with the C-terminus embedded into the bilayer instead of the Nterminus, er and tgn. In the er run, the peptide bound vertically to the membrane for 40ns with the N-terminus interacting with the membrane and then detached from the bilayer. It remained in the solvent for 300ns, then bound again with its C-terminus interacting with the bilayer and remained as such until the end of the simulation. In the second case, as shown in Fig. S5A, the peptide first bound vertically with its N-terminus interacting with the lipids. After 400ns of interaction, it lied horizontally on the surface of the membrane for nearly 230ns. Then, it returned to the original vertical orientation but detached from the membrane and returned to the solvent 950ns into the trajectory. During this time, the non-polar face of the peptide was not directly exposed towards the membrane core, so its bulky hydrophobic residues were not able to interact with the membrane hydrophobic core. After the first 1.5 µs of simulation, the peptide 13 ACS Paragon Plus Environment


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approached the membrane and bound vertically with its C-terminus interacting with the membrane; it remained there until the end of the simulation. This vertical C-terminus conformation, however, lacks physical meaning since the C-terminus of the peptide connects it to the full protein. The analysis for the bound peptide in this conformation are reported separately in subsequent sections simply for reference. Binding mechanism Two initial orientations of the peptide above the bilayer were examined only on the studies with a PC-PS membrane to ensure unbiased results. In either case, the peptide was at least 8 Å above the bilayer, and there was enough solvent to allow peptide diffusion in the water. The polar face of the peptide pointed towards the membrane in the parallel-oriented case (systems labeled with an “o” in Table S2), while the vertically-oriented peptide had its N-terminus closer to the bilayer (labeled with a “z” in Table S2). The initial peptide coordinates were taken from PDBID: 1zhz, with the main helix already folded. In most systems the N-terminus was the first to approach the bilayer, even after detaching from a leaflet and wondering in the solvent. More specifically, the Ser-rich region in the N-terminus, Ser6-Ser9, was the first to make contact with the bilayer. Among all the systems that exhibited stable peptide binding, 18 out of the 21 systems examined, the peptide approached the bilayer with its N-terminus within the first 130-350ns of simulation in 60% of instances. This was particularly true for the znpt and znvt systems, including the one that show no stable binding in the full trajectory (znpt1). Arguably, the Nterminus approached the bilayer first because in these systems the peptide was positioned originally above the bilayer with its main axis aligned to the bilayer normal, the z-axis, and the N-terminus was closest to the membrane. However, the first contact with the bilayer in these 14 ACS Paragon Plus Environment

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systems did not last more than 50ns, after which the peptide returned to the water and interacted with either leaflet of the bilayer due to PBC. Ultimately, the final bound conformation in the znpt and znvt systems, as well as the other systems examined in this study; was not determined by the initial orientation of the peptide. 50% of the systems that started with a vertical peptide above the bilayer resulted in a horizontally bound peptide at the end, and in almost all systems the peptide returned to the solvent and permanently bound the opposite leaflet with respect to its initial position at the beginning of simulation. No direct horizontal binding was observed in the systems with the ER and TGN models due to, apparently, lack of large enough packing defects on the membrane surface. Nearly all these trajectories had the peptide initially bound to the membrane in a vertical conformation that further stabilized into the horizontal conformation (five out of the nine systems). As observed in Fig. 2E, TYR4 is a stabilizing residue of the vertical bound conformation as its ring lays flat near the phosphate region of the lipids. LYS15 and SER18 also contribute to the stability of this conformation interacting with the lipid headgroup atoms, as shown by their individual interaction energies (Table S3). In this conformation, LYS15 interacts with some lipid headgroups, but not to the extent of influencing their lateral organization. On the other hand, the horizontal conformation was stabilized by the interaction of LYS15, TRP10, THR11, and the SER residues in the N-terminus (SER6-9) with the bilayer. In this case, the N-terminus approaches the bilayer, and LYS15 interacts more closely with the lipid headgroups through electrostatic interactions as the main helix binds the membrane. As shown in Figure 2F, the final position of the peptide favors these electrostatic interactions orienting the embedded LYS15 residue towards negatively charged lipids (shown in red), while neutral lipids (shown in cyan) partition to the opposite side. The effect of the ALPS, and amphipathic peptides



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in general, on the lateral diffusion and local distribution of lipids remains to be further explored. Table S4 lists other residues that also interact significantly with the bilayer as determined from hydrogen bonding analysis between the peptide and the membrane on VMD with the following hydrogen bonding criteria: an angle of 30o and a bond length of 3.2 Å. Residues 6-9 in the Nterminus, consecutive serines, are recurrent across all models. Based on hydrophobic interactions, PHE13 and TRP10 are bulky residues that stabilize peptide-membrane interactions when they are embedded into the membrane core - a characteristic of ALPS motifs. THR11 is another bulky residue that stabilizes peptide-membrane hydrophobic interactions and it is located at the interface between the polar and non-polar faces of the peptide; it could also drive rotation of the main helix once this is bound to the membrane by enhancing Hydrogen bonding and electrostatic interactions with lipid headgroups and solvent through its carboxylic acid group. Structural and orientational changes of the peptide during membrane binding The helical character of ALPS changed as it bound to a membrane. Some trajectories show a loss of the α-helix secondary structure to enable proper interactions of non-polar or charged residues and the membrane as seen in the time series in Fig. S10-S11. It is known that ALPS motifs in the unfolded state fold as they bind a membrane through insertion of their bulky hydrophobic groups into packing defects.10,

20

Even though all our simulations started from a

peptide configuration in which the main helix is fully folded (residues 8-18), the 20er1 system unfolded as it approached the bilayer and refolded at the membrane interface, recovering a helical secondary structure. Time series for the RMSD of the main helix is shown for each system in Figs. S12 & S13. Note that in systems znpt (Fig. S12.D), tgn (Fig. S13.D), and 20er1 (Fig. S13.G), the peptide unfolded partially but recover its original conformation after binding


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the membrane; stable binding is shown by the clear decrease in interaction energy between the peptide and the membrane in the corresponding panels in Fig. S14.

Figure 3 – Helix unfolding and binding, example from system 20er1. Time series of the (A) RMSD of the main helix of the peptide; (B) The rotation of the non-polar face of the main helix about its main axis; (C) The helical character of the main helix; (D) Interaction energy between the peptide and the membrane. (E) Snapshots of the peptide interacting with the bilayer; charged lipids are shown in red and neutral lipids in blue for the stably bound leaflet, while lipids from the opposite leaflet are shown with white spheres. (F) Snapshots showing the main helix refolding at the membrane surface prior to stable binding (peptide penetration into the bound leaflet).



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System 20er1 is discussed here to illustrate the refolding of the peptide as part of the binding mechanism. In Fig. 3, the peptide initially approached one of the leaflets horizontally with its hydrophobic residues towards the water (Fig. 3E), but returned to the solvent. The main helix is fully folded, but within the first 100ns the last turn of the helix partially unfolds (Fig. 3A and 3E) and loses helical character (Fig. 3C). Around 300ns, the N-terminus interacted with the other leaflet through electrostatics, but there was not enough space for the peptide to bind permanently and it returned to the solvent and later interacted again with the opposite leaflet. Around 750ns, the peptide approached its final binding leaflet with the hydrophobic residues pointing towards the water, but the last turn of the main helix further unfolded and extended towards the water (Fig. 3C and 3F). To favor electrostatic and hydrophobic interactions with the bilayer, the nonpolar face of the peptide rotated to interact with the bilayer core (Fig. 3B), and the main helix folded correctly restoring its α-helicity; this conformation was retained until the end of the simulation. The helical character did continue to change, as shown by the large RMSD in Fig. 3A, but the main change once the peptide was stably bound to the membrane was the alignment of the rings of Trp10 and Phe13 (Fig. 3.F). The refolding of the main helix, the orientation of the hydrophobic residues towards the bilayer core, and the alignment of the rings of bulky residues in the main helix stabilized this bound conformation, as shown by the decrease of interaction energy in Fig. 3D. Another interesting case was the 20er system, in which the N-terminus of the peptide, usually a random coil, also folded into a helical structure after stable binding was stablished; as a result the fraction of helical character for the full peptide is the highest for this system (Fig. S11E-F). The increase in helical character occurred after 500ns, at the same time that the interaction energy decreased for this system (Fig. S14). Note that the main helix did not unfold in this case, 18 ACS Paragon Plus Environment

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as seen in its RMSD time series (Fig. S13F), and the added turns to the helix did not enhanced the interaction with the membrane, i.e. there was no further decrease in the interaction energy. The rotation of the longitudinal axis of the peptide’s main helix allowed tracking of peptide changes during the binding process. The tracking of the rotation angle was used to show hydrophobic interactions stabilize peptide binding, and the preferred orientation of the non-polar face is towards the bilayer. These results are presented in terms of the absolute value of the cosine of the rotation angle, as presented in Fig. S1. The time series of this rotation angle is shown in Fig. S2-S4 for each system; the data prior to stable contact with the bilayer is mainly noise, given the peptide is free to rotate in water. Upon first contact with the bilayer, the helix still moves on the membrane surface or even detaches from the bilayer prior to stable binding, which adds to the noise in the time series of the rotation angle. In addition, the ability of the main helix to rotate as the peptide moves laterally and the flexible C-terminus coil results in noisy time series even once the peptide is bound in its final conformation. Nonetheless, from these plots, it is clear that the non-polar face of the peptide and its hydrophobic side chains prefer to be oriented towards the bilayer in both the vertical and horizontal conformations. It is important to note that in the vertical conformation the peptide still shifts its position, so the bound structure is at an angle with respect to the membrane surface as presented in the next section and shown in the snapshots of the final orientation of the peptide in Fig. S2-S4. Final bound state of peptide We compute the tilt angle of the main helix with respect to the membrane surface in the final bound conformation of each system. The value was averaged for at least 200ns and blocked every 20ns; results in Table 1 were averaged among systems sharing a bound conformation, and



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the values for individual runs are reported on Table S5. As expected, the tilt angle was smaller for the horizontal binding conformations.

Table 1 – Tilt angle of the main helix with respect to the membrane surface upon stable binding. Angles were averaged among systems that shared the same bound conformation. H=horizontal; V=vertical with the N-terminus embedded into the bilayer; VC=vertical with the C-terminus embedded into the bilayer Conformation PC-PS-V PC-PS –H yeast-V yeast-H yeast-VC

Tilt angle (o) 39.62 ± 4.34 14.73 ± 2.19 36.81 ± 2.69 13.28 ± 3.84 40.58 ± 7.38

The vertical and horizontal conformations differ in the extent of peptide penetration into the bilayer, as does the nature of the membrane in terms of lipid diversity. From Fig.4, and the frequency of contact (FOC) analysis of individual runs presented in Fig. S15 and S16 for the phosphate and glycerol regions respectively, it is clear the peptide is embedded deeper into the complex yeast models. The peptide did not detach after stable binding was stablished, but residues interacted with the bilayer to different extents during the remainder of the trajectory. Notice that from the FOC, each residue is at most about 50% of the time in contact with either the phosphate region, or nearly 20% of the time as deep as the glycerol region. As expected from the orientation of the binding conformations, there is more frequent interaction between the Nterminus of the peptide, especially the SER residues, and the membrane. This is particularly true for the yeast models, in which the peptide is located deeper inside the leaflet.


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Figure 4 - Frequency of contact (FOC) between each residue in the peptide and: (A) the phosphorus atoms of the interacting leaflet; (B) the second carbon of glycerol in lipids of the interacting leaflets. FOC was evaluated within 5Å of the peptide’s Cα atoms. The top horizontal axis lists the residue names and the bottom one the residue sequence numbers. The interaction of the peptide with individual lipid species is shown in Fig. S17; notice that for the simple membrane models, the peptide spends nearly the same amount of time with DOPC or DOPS lipids despite the 60:40 ratio of these species respectively. This would suggest the peptide prefers to position itself at the interface of DOPC-DOPS clusters, as seen in Fig 2.F, which would allow Lys15, a positively charged residue, to interact with the negatively charged PS lipid headgroups. On the other hand, when in contact with complex bilayers, ALPS prefers to interact with PC and PI lipids that constitute nearly 70% of each membrane model taken together. Thus, the frequency of interaction with these lipid species may be rather related to their relative amount with respect to the rest. The fact that the interaction of the peptide with other charged lipid species is not as high, like PS and PA, suggests the main interactions with the bilayer are not driven by electrostatics but are more related to how the side chains interact with the bilayer core. In the context of the full Osh4 protein, this is not surprising since most of the interactions with anionic lipids take place at other regions of the protein and not the ALPS



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motif.29 Additionally, the peptide is embedded more deeply into the TGN membrane model shown by its interaction with ERG molecules. In general, when the peptide is bound in the horizontal conformation, the frequency of interaction with a given lipid species is higher than when bound in the vertical conformation. In all cases, the N-terminus is embedded into the bilayer and penetrates past the phosphate region into the glycerol region of the lipids in the binding leaflet, also shown in the EDPs in Fig. S6-S9, acting as a pseudo-anchor for the peptide. It is not necessarily a true anchor for this motif because some of the simulations did show the N-terminus extended towards the water once the peptide was stable in the horizontal conformation. In addition, studies with the full Osh4 protein, showed SER8 interacting frequently with the bilayer (Fig. 5 in ref 4), although the N-terminus of ALPS extended towards the water. When the full protein is present, the ALPS motif does not penetrate into the hydrophobic core, but it interacts with lipids only at the surface. It is likely that the timescales were too short in that study to capture the extent of interaction and penetration of ALPS motif into the bilayer. Binding energetics between peptide and membrane The energetics between the peptide and each bilayer as well as the contribution of individual residues was probed to obtain an estimate for the enthalpic contribution to binding; which contributes partially to the overall binding free energy and should not be taken as a sole indicator of the strength of binding. Table 2Error! Reference source not found. summarizes the blocked averages of interaction energies for systems sharing a bound conformation along with the contribution of the main helix. The contributions of each residue for the vertical and horizontal conformations are depicted on Fig. 5, and Table S3 lists the values of interaction energies for each residue in individual runs. The time series of the interaction energy for each system are shown in Fig. S14, from which one can determine the time stable binding was stablished as the time when the energy decreases. As anticipated, the horizontally bound conformation is enthalpically more stable as it results in the non-polar side changes of the main helix pointing towards and interacting with the membrane hydrophobic core. The energy contribution of the main helix to the total interaction energy constitutes nearly a third for the vertical conformation, but it is nearly half of the total energy in the horizontal conformation. The equilibrium binding 22 ACS Paragon Plus Environment

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energy of the vertical conformation is, in all cases, nearly half that of the horizontal conformation. However, there is no statistical difference between the binding energies of each conformation with respect to the membrane model (PC-PS vs. yeast).

Figure 5 - Interaction energy for each residue in the peptide computed over at least 500ns of equilibrated trajectory after binding is stablished; charged residues are marked with a ‘*’. The red box delineates the residues in the main helix. In both membrane models, PC-PS and yeast, Tyr4 and Lys 15 are large contributors to the interaction energy of the peptide in the vertical and horizontal conformation, respectively (see also Fig. 2). Additionally, Lys15 interacts directly with the membrane in the yeast-c bound conformation, which is not biologically relevant since the C-terminus of ALPS is attached to the full Osh4 protein and could not embed into the bilayer. The residue names are listed on the top horizontal axis, and the bottom one lists their number. Table 2 – ∆Ebind between ALPS and model membranes in the vertical (V=N-terminus interacting with bilayer, VC=C-terminus interacting with bilayer) and horizontal (H) conformations. Averages and standard error are reported among systems sharing a bound conformation. Conformation PC-PS-V PC-PS-H yeast-V yeast-H yeast-VC

∆Ebind (kcal/mol) full peptide main helix -60.46 ± 14.21 -22.24 ± 4.72 -124.17 ± 9.59 -70.59 ± 5.22 -67.58 ± 16.71 -23.06 ± 7.19 -129.57 ± 12.96 -65.09 ± 4.62 -112.44 ± 18.74 -74.03 ± 9.66

The use of surface tension to effectively create more packing defects on the membrane surface did influenced the binding strength of the final bound conformation among systems that share that final state (Table S6). The trend was consistent for the horizontal conformation in both the PC-PS and the yeast models; the binding strength increased for the simulations with imposed 23 ACS Paragon Plus Environment


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surface tension 20% and 30.5% per membrane model respectively, with respect to the models run without added surface tension. The same was true for the vertical conformation among the systems run with the yeast membrane models. However, for the runs with the PC-PS membrane model that resulted in a vertically bound peptide, the binding strength decreased nearly 50% with respect to the systems run without surface tension. We did not examine the free energy landscape of peptide binding, however our observations suggest the vertical binding conformation could be an intermediate state that leads to, but is not required for, the horizontal conformation. In most of the yeast membrane models we observe the peptide bound the bilayer in a vertical or tilted conformation before transitioning to the horizontal state, yet we never observe a transition from the horizontal to the vertical conformation. The same is the case in a separate study we conducted to examine the relationship between packing defects and binding events using the highlymobile membrane-mimetic (HMMM) model.62 On the other hand, the vertical conformation is not an unstable state, as systems in which the peptide bound in this conformation did not unbind and remained in this conformation for over 850 ns in both the DOPC-DOPS and the yeast membrane models, even in the systems run with surface tension.

Additional features There are three interesting cases worth mentioning individually, two PC-PS systems and one of the yeast models. For the znvt1 and znvt2 systems (Fig. S14), there is a further decrease in the interaction energy when the non-polar face of the main helix rotates to point towards the bilayer core, both systems had a final horizontally bound conformation. For znvt1, shown in Fig. 6, the peptide approaches the bilayer and binds through its N-terminus and the main helix lays horizontally on the membrane surface. Initially, the non-polar face points towards the water (Fig. 6.C), the peptide remains bound to the bilayer until the end of the simulation, but oscillates between a vertical and a horizontal bound conformation until the main helix rotates and the non-


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polar residues point towards the hydrophobic core of the membrane. After this, the peptide remains in the horizontal conformation and embeds below the lipid headgroup region. For znvt2, the peptide initially bound the membrane vertically nearly 400 ns into the trajectory; then it tilted towards the horizontal conformation after 600 ns, where a decrease in interaction energy is observed (Fig. S14D). Finally, the tgn yeast model, discussed in detail at the end of the “Binding conformations” section, bound firmly to the membrane several times for periods longer than 200ns but in three different conformations (Fig. S5A). The interaction energy profile for this system (dark line in Fig S14G) shows the horizontal conformation is energetically favored, but it is not stable unless hydrophobic interactions of the non-polar face can be stablished with the bilayer core and there is enough space for the peptide to penetrate below the lipid headgroup region. From visual examination of this trajectory, the horizontally-bound peptide of the tgn system had its non-polar face looking towards the water (Fig. S5A.ii), which resulted in unbinding of the peptide after approximately 230ns of contact.



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Figure 6 – Binding mechanism steps for ALPS with the znvt-1 membrane model. (A) Interaction energy between peptide and membrane. (B) Rotation of the non-polar face of the main helix about its main axis, refer to Fig. S1 for the meaning of the cosine values. (C) Snapshots of the peptide corresponding to the times indicated by the red lines in panels (A) & (B)

Discussion From our studies on the binding mechanism of the ALPS motif of Osh4, we see that bulky residues like PHE and TRP, stabilize the hydrophobic interactions of the peptide as is characteristic of this type of AHs.9, 22 THR, also a bulky residue, stabilizes peptide-membrane interactions, but its hydroxyl group can also contribute to hydrogen bonding and to the rotation of the main helix so its non-polar face is pointed towards the bilayer. Presence of large lipid packing defects does facilitate the initial survey of the peptide at the membrane surface; however, it may not be determinant for stable peptide binding. Even for the two systems in this study that did not show permanent peptide binding despite several superficial contacts, the peptide interacts with the lipid headgroups at most 80 ns at a time. Given binding is a stochastic process such variability in binding events was anticipated. Simulations with the more complex yeast membrane models showed that if ALPS approaches the bilayer in an orientation that allows the interaction of residues in its N-terminus – particularly TYR4 or the sequence of SER6-SER8 – with a packing defect, even a small one, the peptide remains attached to the bilayer as it unfolds and binds firmly to the membrane refolding into an AH and creating a larger defect. The anionic character of a bilayer is a factor that influences the binding timescale of the peptide. ALPS bound, in general, within the first 300-700ns of simulation to the PC-PS and TGN models, both of which that contain between 40-43% of anionic lipids. In contrast, the ER model has only 35% anionic character, and the peptide permanently bound to the ER membranes after 1µs of simulation in most cases, and only once about 500ns into the trajectory. The ER and TGN membrane models are very similar in terms of surface area per lipid and compressibility 26 ACS Paragon Plus Environment

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modulus; in fact, the order of lipid tails is very similar between the two as well, as was presented in a previous study.36 Besides the anionic character, a key difference between them is the unsaturation degree of the lipid tails, 0.69 for the ER and 0.74 for the TGN. In all cases the peptide did bind and unbind before acquiring its final stable conformation; clearly the membrane properties influence the extent and strength of interaction between peptide and membrane. In general, the ALPS peptide preferred to interact with membranes with higher anionic character and with higher unsaturation degree (both the TGN and PC-PS models). Membrane exploration or surveying took place between 400 and 900 ns depending on the system, nearly 1.3 µs for the tgn system in which the peptide bound the membrane in different conformations for several nanoseconds and then returned to the solvent (see Fig. S5.A). Membrane surveying took longer, on average, for the systems that resulted in a vertically bound peptide; a summary of the contact times between the peptide and the membrane is provided in Table S7 for reference to the reader. Just as the membrane character influences peptide binding, the presence of the peptide in the leaflet to which it binds also affects the local membrane structure. Upon binding, ALPS is able to rotate about its main axis to stabilize the bound conformation, especially if its non-polar face is only partially in contact with the membrane hydrophobic core. This discussion is also at the center of current research, along which our results suggest there is a feedback between peptide binding and folding, and packing density recognition, curvature inducing effect, and local lipid composition as well as electrostatics.9, 18, 24, 63 No preference was shown for a given bound conformation (vertical vs. horizontal) based on the initial orientation of the peptide above the bilayer. In all cases, SER8 was a recurring interacting residue and usually the first one to approach the bilayer along with other SER residues in the N-terminus of the peptide. Although peptides bound in the vertical conformation



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remained bound until the end of the simulation, the horizontal conformation is favored energetically and shows a more interesting binding mechanism that aligns better to what has been reported for other AHs.6, 9 In fact, a horizontal conformation in which the non-polar face of the peptide is oriented toward the membrane core displays a further decrease in the interaction energy, i.e, a more stable conformation, as it was mentioned in the precious section. Looking at the bigger picture, i.e. including the entire protein (Osh4), the peptide plays rather a surfacescanning role and it does not permanently or strongly bind to the membrane.4 ALPS seems to act as a sensor rather than an anchor to stabilize Osh4-membrane interactions. There is further need to characterize ALPS-membrane interactions in the presence of the full protein, especially when Osh4 is in the proper conformation to enable lipid uptake and subsequent transport. In the literature, there is still debate on whether curvature sensing AHs are also lipid packing sensors, or if one implies the other.9,

23, 64-66

Nonetheless, there is consensus in the effect of

ALPS, and AHs, binding to membranes and reducing the stress caused by lipid shape mismatch and inherent curvature.19, 67 Fig. 7 shows two sample systems, one for each bound conformation, and the relative size of the packing defect below the peptide after stable binding was stablished. Simply considering the penetration depth of the peptide and its projection on the membrane plane for the vertical and horizontal conformations, it is obvious the final size of the packing defect will be larger for the peptide bound horizontally. The question of whether the peptide bound a membrane because was a large enough packing defect, or if the peptide itself created the packing defect upon binding is more complex and requires a cause-effect analysis. We saw both cases among the systems included in this study, and this aspect is studied in a separate publication.62


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Figure 7 – Lipid packing defect below a bound peptide in the (A-B) vertical and (C-D) horizontal conformations. The hydrophobic lipid tails are shown in white, and lipid head groups are shown in red with nitrogen atoms in blue.

Conclusion We studied the binding mechanism of the ALPS motif of Osh4 in yeast using all-atom molecular dynamics. Through these studies, we examined the influence of membrane structure and composition on peptide binding events. From the overall 42µs of simulations, we determined two biologically relevant bound conformations. A vertical one with the N-terminus of the peptide embedded into the membrane, and a more favorable horizontal conformation with the hydrophobic side chains of the peptide’s main helix embedded into the hydrophobic core of the membrane. The binding strength is higher for the horizontal conformation in both the PC-PS and 29 ACS Paragon Plus Environment


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the yeast membrane models (2 and 9+ lipid types, respectively). Our studies show that membrane structure and lipid character influence peptide interaction with the bilayer. The ALPS motif is slightly more attracted to membranes with higher anionic character, like the PC-PS and TGN systems (40% and 42.7% anionic character, respectively), than to the ER membranes (37% anionic character). The N-terminus, rich in serine residues, is in contact with the bilayer in nearly all the bound instances, and its SER region (residues 6-9) is usually the first to interact with the bilayer in transient contacts before stable binding. Bulky residues like TRP10 and PHE13 in the main helix stabilize the horizontal conformation, and electrostatic interactions of LYS15 also contribute to stable binding as shown by its large contribution to the interaction energy (Fig. 5). Moving forward, there is still need to examine the influence of natural membrane curvature on peptide binding, which may be important beyond the task of generating packing defects and more related to lipid diffusion and the reorganization of lipids. Understanding the feedback loop between ALPS binding and packing defects as well as the resulting local changes in lipid composition at the binding site will contribute to better understanding of peripheral membrane protein mechanisms and regulation. Supporting Information The following material is available as supplementary data and has been referenced throughout this work: tables listing the components and size of individual systems, time of peptide contacts with the bilayer as well as bound conformation, interaction energies of the full peptide and per residue with the bilayer, hydrogen bonding between peptide and membrane, and tilt angle of the bound peptide with respect to the membrane surface. Figures provided include: a diagram depicting the interpretation of the helix rotation angle upon its main axis, time series and representative snapshots of the aforementioned helix rotation, electron density profiles showing


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the relative position of the peptide with respect to the lipid headgroups at the beginning and end of each simulation trajectory, bar plots showing the frequency of contact between each residue and the membrane lipids, and time series for the helical character of the peptide as it bound the bilayer, its RMDS with respect to its initial configuration, and interaction energy with each bilayer. Acknowledgements This study was funded in part by NSF grants DBI-1145652 and MCB-1149187 the High Performance Deepthought & Deepthought 2 Computing Clusters at the University of Maryland, College Park supported by Division of Information Technology. Anton computer time was provided by the Pittsburgh Supercomputing Center (PSC) through Grant R01GM116961 from the National Institutes of Health and our specific time associated with the grant PSCA14030P. The Anton machine at PSC was generously made available by D.E. Shaw Research. References. 1. Stryer, L., Molecular Design of Life. W.H. Freeman and Company: New York, USA, 1989. 2. Bassereau, P.; Jin, R.; Baumgart, T.; Deserno, M.; Dimova, R.; Frolov, V. A.; Bashkirov, P.; Grubmüller, H.; Jahn, R.; Risselada, H. J., et al., The 2018 biomembrane curvature and remodeling roadmap. Journal of Physics D: Applied Physics 2018, 51 (34), 343001. 3. van Meer, G.; Voelker, D. R.; Feigenson, G. W., Membrane lipids: where they are and how they behave. Nat Rev Mol Cell Biol 2008, 9 (2), 112-24. 4. Monje-Galvan, V.; Klauda, J. B., Peripheral membrane proteins: Tying the knot between experiment and computation. Biochim Biophys Acta 2016, 1858 (7 Pt B), 1584-93. 5. Doucet, C. M.; Esmery, N.; de Saint-Jean, M.; Antonny, B., Membrane Curvature Sensing by Amphipathic Helices Is Modulated by the Surrounding Protein Backbone. PLoS One 2015, 10 (9), e0137965. 6. Antonny, B., Mechanisms of Membrane Curvature Sensing. Annual Review of Biochemistry, Vol 80 2011, 80, 101-123. 7. D'Angelo, G.; Vicinanza, M.; De Matteis, M. A., Lipid-transfer proteins in biosynthetic pathways. Curr Opin Cell Biol 2008, 20 (4), 360-70. 8. Jackson, C. L.; Bouvet, S., Arfs at a Glance. Journal of Cell Science 2014, 127 (19), 4103-4109. 9. Vanni, S.; Vamparys, L.; Gautier, R.; Drin, G.; Etchebest, C.; Fuchs, P. F.; Antonny, B., Amphipathic lipid packing sensor motifs: probing bilayer defects with hydrophobic residues. Biophys J 2013, 104 (3), 575-84. 31 ACS Paragon Plus Environment


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TOC Graphic


Preferred Binding Mechanism of Osh4's Amphipathic Lipid-Packing

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