# Structural and dynamics of apoA-1 mimetic peptide lipid nanodisc assemblies: A molecular dynamics study

Rohith Ravi, Evgeniy S Salnikov, Burkhard Bechinger, Mounir Tarek

► To cite this version:

Rohith Ravi, Evgeniy S Salnikov, Burkhard Bechinger, Mounir Tarek. Structural and dynamics of apoA-1 mimetic peptide lipid nanodisc assemblies: A molecular dynamics study. Biochimica et Biophysica Acta:Biomembranes, 2025, 1868 (2), pp.184495. 10.1016/j.bbamem.2025.184495. hal-05464583

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Research paper

# Structural and dynamics of apoA-1 mimetic peptide lipid nanodisc assemblies: A molecular dynamics study

Rohith Ravi a,*, Evgeniy S. Salnikov b,*, Burkhard Bechinger b,c, and Mounir Tarek a,†

## ARTICLE INFO

### Keywords:

All-atom molecular dynamics simulation

Apolipoprotein-A1 mimetic peptides

Lipid nanodiscs

Solid-state NMR

## ABSTRACT

Apolipoprotein A-I (apoA-I) mimetic peptides, inspired by the principal protein component of high-density lipoprotein, self-assemble with lipids to form discoidal nanodiscs widely used in biomedical research and as versatile scaffolds for characterization of membrane proteins in structural biology. Here, we investigate the 14A apoA-I mimetic, quantifying its orientation around the lipid bilayer and identifying the interactions that are crucial for nanodisc stability and dynamics using all-atom molecular dynamics simulations. To assess model fidelity, we back-calculated solid-state NMR observables, namely ¹⁵N chemical shifts and ²H quadrupolar splittings from the trajectories and compared them with previously reported solid-state NMR data. The simulations support a dimeric, antiparallel, belt-like arrangement of 14A peptides around the discoidal bilayer, stabilized by π-π stacking between aromatic residues and by electrostatic and hydrophobic peptide-lipid interactions. These interactions yield structurally stable nanodiscs with pronounced heterogeneity in lipid ordering and bilayer thickness between the nanodisc center and rim. Collectively, our MD results provide atomistic evidence for previously hypothesized peptide-peptide and peptide-lipid interactions and clarify how amphipathic helices organize to form the rim of discoidal nanodiscs. These insights inform the rational design of apoA-I mimetics for biomedical applications and the optimization of nanodiscs as platforms for studying membrane proteins.

## 1. Introduction

Lipid nanodiscs are discoidal supramolecular complexes in which a lipid patch is stabilized by amphipathic α-helical polypeptides [1]. A physiologically significant component of these structures is nascent high-density lipoprotein (nHDL), which plays a critical role in reverse cholesterol transport (RCT) and serves as a protective factor against the progression of cardiovascular disease (CVD) [2–4]. nHDL formation occurs during the initial stages of RCT when amphipathic α-helical apolipoprotein A1 (APOA1) interacts with the ATP-binding cassette transporter A1 (ABCA1). ABCA1 facilitates cholesterol and lipid efflux from macrophages, maintaining cellular cholesterol homeostasis essential for proper cellular function [5–7]. Genetic mutations or defects in ABCA1 lead to Tangier Disease [8], which causes impaired nHDL formation and maturation, resulting in cholesterol accumulation within arterial walls. This accumulation promotes plaque formation, increasing the risk of developing atherosclerosis.

Attempts to artificially elevate nHDL levels have been explored [9]. However, the clinical studies conducted in animal models, and to a

certain extent in humans, indicate that merely increasing nHDL does not consistently deliver the anticipated cardioprotective effects [10]. Consequently, there has been growing interest in developing APOA1-mimetic peptides capable of forming synthetic HDL (sHDL). These peptides are designed using APOA1 as a structural template and have been extensively evaluated for lipid-binding affinity, anti-inflammatory effects, and overall atheroprotective properties [11–13]. Notably, a phase 1 clinical trial involving these peptides demonstrated promising improvements in HDL quality in vivo, underscoring their therapeutic potential [14].

These peptide-lipid complexes can serve as an alternative platform [15,16] for biophysical characterization of membrane proteins (MPs), due to the complex's structural and functional roles [17] and the advantage of providing a detergent-free bilayer environment. Approximately 50% of all pharmaceutical drug targets the membrane proteins [18], highlighting the significance of such characterization platforms. Conventional membrane scaffold proteins (MSPs), such as APOA1-based MSPs, are covalently linked via glycine or proline

<footnote>a Université de Lorraine, LPCT, CNRS, UMR7019, 54000 Nancy, France</footnote>

<footnote>b Institut de Chimie, Université de Strasbourg, CNRS, UMR7177, 67000 Strasbourg, France</footnote>

<footnote>c Institut Universitaire de France, 75005 Paris, France</footnote>

<footnote>* Corresponding author.
E-mail address: mounir.tarek@univ-lorraine.fr (M. Tarek).</footnote>
residues, creating discs with fixed diameters typically ranging from 10 to 17 nm [19–21]. By contrast, engineered APOA1-mimetic peptides form nanodiscs through non-covalent interactions, allow size modulation by adjusting peptide-to-lipid ratios [22,23]. Hence, the amphipathic peptides mimic the role of MSPs [24] and stabilize the discoid lipid patches [23,25].

Despite extensive research on nanodiscs for membrane protein studies and the medicinal importance of sHDL peptide-lipid complexes, the structural and physicochemical properties of these systems remain inadequately explored. Most APOA1-mimetic peptides studied in vivo derive from the class A amphipathic helix peptide “18A,” [24,25], an 18-amino acid peptide often modified through targeted amino acid substitutions to enhance functionality [26–28].

In this work, we focused on a shorter variant of the amphipathic α-helical 18A peptide, the 14A peptide, obtained by removing one helical turn. This truncation simplifies synthesis and stable-isotope labeling for NMR studies (sequence details are provided in Section 2.1). Previously reported NMR studies on 14A-based nanodiscs have shown that they provide a bilayer environment compatible with the characterization of membrane proteins. Notably, these 14A peptide-based nanodiscs can be tuned up to ≈30 nm in diameter, well align with the applied magnetic field, and serve as an alignment medium for measuring residual dipolar couplings (RDCs) of membrane proteins without detergents [23].

Additionally, Salnikov et al. [27] examined the structural aspects of 14A-peptide-based nanodiscs and provided robust experimental evidence that the peptides adopt an orientation parallel to the plane of the discoidal lipid patch. Site-specific labeling and solid-state NMR (ssNMR) measurements further indicated that these peptides localize at the nanodisc rim and possibly form head-to-tail, antiparallel dimers, similar to those in apolipoprotein A-I-lipid complexes [27,29]. However, the detailed atomistic arrangement of the amphipathic helices that form the rim around the discoidal lipid patches, as well as the peptide-lipid and peptide-peptide interactions that stabilize the nanodiscs, remain unexplored.

To address this gap, we employ all-atom molecular dynamics (MD) simulations to study lipid bilayer patches composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DMPC) stabilized by 14A peptides. MD simulations were conducted at 30 °C and 37 °C for two different-sized nanodiscs (Table 1), and the resulting peptide orientations were compared to existing solid-state NMR data acquired at 37 °C [27]. This comparison allows us to assess how temperatures near and below the experimental condition, particularly in relation to the DMPC phase transition and peptide thermal fluctuations, influence peptide orientation, thereby helping to validate the accuracy and transferability of our simulation model.

We further investigate the physicochemical properties responsible for the peptide dimer's stabilization and examine how peptide-lipid interactions and the size of the nanodisc influence lipid behavior. Our comprehensive analysis delivers an atomic-level characterization of nanodiscs formed by 14A peptides and DMPC lipids, offering critical information relevant to their structural and functional stability. These findings ultimately help future design of APOA1-mimetic peptides, guiding the development of more effective therapeutic agents for cardiovascular disease and potentially advancing the quest for an alternative platform for handling membrane proteins.

## 2. Methods and computational details

### 2.1. Peptide of interest

The 18A and 14A peptides have the following sequences:

18A: Ac-DWLKA FYDKV AEKLK EAF-NH₂

14A: Ac-DYLKA FYDKL KEAF-NH₂

### 2.2. Computational details

Atomistic molecular dynamics simulations of apoA-I mimetic 14A/DMPC nanodiscs were conducted following a systematic protocol. Peptide monomers were initially generated using PEP-FOLD [30], with subsequent N- and C-terminal modifications by acetylation and amidation performed through CHARMM-GUI [31] (Fig. S1).

Based on previous NMR studies by Salnikov et al. [27] of 14A peptides and other related apo A1 mimetic peptides [25,26], we hypothesized that the 14A peptides form an antiparallel, head-to-tail dimer. To obtain initial dimeric configurations that both stabilize the nanodiscs and are consistent with the ssNMR constraints, we explicitly tested antiparallel head-to-tail and parallel 14A dimer arrangements. A full description of these tests and refinements, together with the corresponding analysis and figures, is provided in the Supporting Information (Section 1). We obtained the peptide dimeric conformation that met our structural hypothesis (an antiparallel dimeric arrangement and compatible with rim localization), and we used it as a template to assemble the 14A peptide-based DMPC nanodisc (Fig. S4(ii)).

The optimized anti-parallel dimeric peptide arrangement was used to pre-assemble an amphipathic belt-like structure. We modeled two different sizes of 14A-based nanodiscs (Table 1) to reflect widely used experimental nanodisc platforms for membrane protein biophysical characterization. The smaller nanodisc, ≈9.8 nm in diameter, corresponds to the typical size formed by MSP1 [1,32], while the larger nanodisc, around 18 nm in diameter, matches the dimensions of MSP2N2-based nanodiscs [1]. These two sizes were selected because they are commonly employed for membrane protein studies.

Depending on the nanodisc size, an appropriate number of energy-minimized dimers was first arranged in a circular ring to form a peptide belt, and a well-equilibrated DMPC bilayer patch was then inserted into the center of this ring. This protocol for constructing peptide-based nanodiscs is conceptually similar to previously reported molecular models of nanodiscs [33]. The resulting peptide-lipid assembly (Fig. S7) was subsequently solvated in an aqueous solution containing 0.15 M NaCl and TIP3P water molecules. All simulations were carried out using the CHARMM36m force field [34] and the GROMACS software suite [35].

Following energy minimization, each system underwent a multi-step equilibration process, beginning with 2 ns simulations at constant temperature and volume, followed by constant temperature and pressure simulations using a 1 fs timestep. During this phase, positional restraints on the peptide backbones and lipid head groups were gradually released to allow system relaxation. Subsequent production MD simulations were carried out with a 4 fs timestep using Hydrogen Mass Repartitioning (HMR) [36] at constant temperatures of either 303.15 K or 310.15 K and constant pressure of 1 bar. Temperature was controlled using the Nosé-Hoover thermostat [37] with a relaxation time $τ_i = 0.5$ ps. Pressure was maintained isotropically at 1 bar, with a compressibility of $3 \times 10^{-4} \text{bar}^{-1}$, using the Parrinello-Rahman barostat [38] with a relaxation time of $τ_p = 4.0$ ps.

The long-range electrostatic interactions beyond 1.0 nm were calculated using the Particle Mesh Ewald (PME) [39] method, and van der Waals interactions were switched to zero for distances larger than 1.0 nm. All the MD simulations were performed under three-dimensional periodic boundary conditions. This protocol ensured the robust construction and simulation of the 14A/DMPC nanodiscs, allowing for a detailed investigation of their structure, dynamics, and stability. In addition, a pure bilayer consisting of DMPC was simulated by applying the same simulation protocol, apart from using the NPT ensemble with anisotropic pressure control. Further, simulation details for each system are presented (Table 1).

### 2.3. Trajectory analysis

All analyses were conducted using GROMACS tools [35] and MD-Analysis [40] unless otherwise stated.
<caption>**Table 1**</caption>

Simulation system details for MD studies of DMPC bilayer patches stabilized by 14A peptides. System A contains 24 peptides (12 peptide dimers), and System B contains 40 peptides (20 dimers). Each system includes DMPC lipids, water molecules, and ions to ensure proper solvation and charge neutrality. For each system, we report the number of peptides, number of lipids, total number of atoms, total simulation time, and the number of replicas at 30 °C and 37 °C.

<table><thead><tr><th>System</th><th>No. of peptides</th><th>No. of lipids</th><th>Total no. of atoms</th><th>Total simulation time (ns)</th><th>Replicas (Temperature)</th></tr></thead><tbody><tr><td>A</td><td>24</td><td>186</td><td>326 485</td><td>1000 × 6</td><td>3 × 30 °C, 3 × 37 °C</td></tr><tr><td>B</td><td>40</td><td>664</td><td>1 447 090</td><td>400 × 2</td><td>1 × 30 °C, 1 × 37 °C</td></tr><tr><td>Pure DMPC bilayer</td><td>0</td><td>154</td><td>38 948</td><td>500 × 2</td><td>1 × 30 °C, 1 × 37 °C</td></tr></tbody></table>

As a preliminary analysis, the radius of gyration ($R_g$) of the nanodiscs was monitored over time to assess whether the system had reached equilibrium, with convergence of $R_g$ serving as an indicator. Additionally, the secondary structure content of the peptides was analyzed using the Dictionary of Secondary Structure of Proteins (DSSP) algorithm [41], as implemented in MDAnalysis. Further analyses focused on peptide-peptide interactions, peptide-lipid interactions, and lipid properties, such as bilayer thickness and lipid order parameters, to provide a comprehensive description of nanodisc stability.

To characterize the peptide-peptide interactions, we calculated free-energy profiles for the most probable intermolecular interactions between the tyrosine (Y7–Y7) and phenylalanine (F14–F14) aromatic rings, independently. For each pairwise interaction, we computed the distance $r$ between the centers of mass of the aromatic rings and the angle $\theta$ between their ring normal vectors. These two geometric parameters together define the relative orientation and proximity of the aromatic side chains, allowing classification of common $\pi-\pi$ interaction motifs: face-to-face stacking, parallel-displaced stacking, and T-stacking (edge-to-face).

Relative Gibbs free energy $\Delta G(r, \theta)$ was estimated through the Boltzmann distribution based on the resulting probability distribution $P(r, \theta)$:

$$ \Delta G(r, \theta) = -k_B T \ln P(r, \theta) \quad (1) $$

where $k_B$ is the Boltzmann constant (in kcal/(mol·K)) and $T$ is the simulation temperature (310 k) in Kelvin.

Peptide-lipid interactions are pivotal for nanodisc stability, primarily through salt bridge formation. We computed radial distribution functions (g(r)) for two atomic pairs: the nitrogen atom of lysine's amino group with the phosphorus atom of DMPC, the carbon atom of glutamate's carbonyl group with the nitrogen atom of choline group of DMPC, and the hydroxyl group of tyrosine with the nitrogen atom of choline group of DMPC. The analysis identified cutoff distances of $\le 4.7$ Å for lysine-phosphorus, $\le 5.7$ Å for glutamate-nitrogen interactions, and $\le 5.7$ Å for the tyrosine-nitrogen, which were subsequently used to determine the average number of salt bridges per peptide (Fig. S11).

Upon visual inspection of the MD trajectory, the aromatic rings of tyrosine (2Y) and phenylalanine (6F) residues were observed to anchor at the bilayer interface, engaging in hydrophobic interactions with the acyl chains. To quantitatively assess this phenomenon, we calculated the angle between the normal to each aromatic ring plane and the normal to the bilayer (Fig. S13). This angle was determined for every peptide over the entire simulation trajectory; the per-frame averages were then computed and plotted as a function of time.

The average diameter of the nanodisc was determined by assuming a discoidal shape [42]. We measured the diameters along the major and minor in-plane axes (x and y, respectively) from the mass density distributions. The mass density distributions of the peptides and the lipid bilayer were calculated and symmetrized around the nanodisc center of mass to ensure consistent measurements along the x- and y-axes.

To obtain an average diameter with associated uncertainties, each production trajectory (Table 1) was divided into non-overlapping 100 ns time blocks. Because gmx density produces a single time-averaged profile over the analysis window, we computed separate mass

density profiles for each 100 ns block. For every block, we then measured the distances from the center to the points on either side at which the mass density distribution decayed to 1 kg/m$^3$. These distances define the block-wise diameters. The reported average diameter is the mean of the block-wise diameters, and the standard deviation across blocks quantifies the uncertainty.

In addition, we characterized key lipid properties, specifically bilayer thickness and the lipid order parameter for both the nanodisc and a simulated DMPC lipid bilayer. We defined bilayer thickness as the minimum distance along the bilayer normal between phosphate headgroup pairs in the two leaflets. These headgroup pairs were identified for each leaflet and stored together with the measured thickness values and their corresponding xy coordinates. The data were subsequently binned into small grids based on the box dimensions in the xy plane, with each bin associated either with the top or the bottom leaflet. An averaged grid was then generated by combining the two leaflet-specific grids.

The order parameter $S_{CH}$ of the lipid acyl chains was calculated according to:

$$ S_{CH} = \frac{1}{2} \left\langle 3 \cos^2(\theta) - 1 \right\rangle, \quad (2) $$

where $\theta$ is the angle between the C-H bond and the bilayer normal. The $S_{CH}$ values were computed over all trajectories for each lipid and each carbon along both lipid tails [43,44]. These values were then averaged across the two tails and binned onto a grid. An additional average was calculated over all frames and lipids within each bin. To further characterize the lipid tail profile, we also performed a global average across snapshots, lipids, and tails. The same methodology was applied to obtain $S_{CH}$ from the DMPC bilayer simulations.

## 2.4. $^2$H-NMR quadrupolar splittings and $^{15}$N chemical shifts from MD simulation

We computed the $^2$H-NMR quadrupolar splittings of the $C_\alpha - C_\beta H_3$ groups of residues Ala5 and Ala13, and the $^{15}$N chemical shifts of residues Leu10 and Ala5, following the same procedures used experimentally [44–47]. These quantities depend on the orientation of the peptides relative to the nanodisc bilayer [48].

For a fair comparison between experimental and MD simulation values, we calculated the experimental-like $^2$H quadrupolar splittings corresponding to the orientation of the $C_\alpha - C_\beta$ bonds in residues Ala5 and Ala13 with respect to the membrane normal [44–46]. In the experiments, the nanodisc normal is aligned parallel to the external magnetic field; accordingly, in our simulations, we oriented the nanodisc normal along the z-axis (Fig. 1A).

The static quadrupolar splittings ($\Delta v_Q$) for each residue at a particular trajectory snapshot is calculated as:

$$ |\langle \Delta v_Q \rangle| = \frac{3}{2} \frac{(e^2 q Q / h) \cdot \frac{3 \cos^2 \theta - 1}{2}}{(e^2 q Q / h)} \quad (3) $$

where $\theta$ is the angle between the $C_\alpha - C_\beta$ bond and the hypothetical magnetic field taken as z axis, and $(e^2 q Q / h)$ is the static quadrupolar coupling constant, taken as 84 kHz, in agreement with experimental NMR measurements on 14A peptides [27,46].
<figure><img src="image_2.png" /><figcaption>Fig. 1. Pictorial representation of the method used to compute $^2$H quadrupolar splitting and $^{15}$N chemical-shift anisotropy (CSA). (A) The $^2$H splitting is obtained from the angle $\theta$ between the $C_a-C_b$ bond vector and the magnetic-field direction $\mathbf{B}_0$. (B) The $^{15}$N CSA tensor is shown in its principal-axis system placed next to the molecular frame of the peptide-bond. $^{15}$N chemical shift is obtained by determining the polar ($\Theta$) and azimuthal ($\Phi$) angles of $\mathbf{B}_0$ within the molecular frame relative to the principal axes of the CSA tensor. Axes $\vec{\sigma}_{11}$ and $\vec{\sigma}_{33}$ lie in the peptide plane within the plane of the paper, while $\vec{\sigma}_{22}$ is perpendicular to it. $\Theta$ is measured between $\vec{\sigma}_{33}$ and $\mathbf{B}_0$, and $\Phi$ is measured between the $\vec{\sigma}_{11}$ and the projection of $\mathbf{B}_0$.</figcaption></figure>

Additionally, we calculated the $^{15}$N chemical shift, following the experimental methodology [44,47,49]. This chemical shift depends directly on the orientation of its tensor eigenvectors relative to the external magnetic field. The eigenvector $\vec{\sigma}_{33}$ is oriented approximately 16° from the N-H bond toward the N-C bond [50], lying in the peptide plane. The eigenvector $\vec{\sigma}_{11}$ is perpendicular to $\vec{\sigma}_{33}$, also within the peptide plane, while $\vec{\sigma}_{22}$ is orthogonal to both, thus perpendicular to the peptide plane. All vectors originate from the nitrogen atom (Fig. 1B).

Defining the Euler angles $\Phi$, $\Theta$, and $\Psi$ to transform from the eigenvector reference frame to the laboratory frame (z axis) [47], the chemical shift component $\sigma_{zz}$ can be calculated as:

$$\sigma_{zz} = \sigma_{11} \sin^2 \theta \cos^2 \Phi + \sigma_{22} \sin^2 \theta \sin^2 \Phi + \sigma_{33} \cos^2 \theta \quad (4)$$

where $\vec{\sigma}_{zz}$ is the $^{15}$N chemical shift aligned with the membrane normal, and the eigenvalues of the chemical shift tensor $\sigma_{11}$, $\sigma_{22}$, and $\sigma_{33}$ are respectively taken as 56 ppm, 81 ppm, and 223 ppm, as used in experimental NMR studies on 14A peptides [27]. Because MD simulations generate discrete snapshots at finite time intervals, the raw distributions of both the static quadrupolar splittings ($\Delta v_Q$) and the $^{15}$N chemical shifts ($\sigma_{zz}$) are intrinsically discontinuous. We therefore applied the kernel density estimation (KDE) implementation from scikit-learn [51] to obtain smooth, continuous approximations of these probability distributions. The mean ($\mu$) and standard deviation ($\sigma$) were then computed from the KDE-derived profiles for direct comparison with experimental values.

# 3. Results and discussions

## 3.1. Preliminary analysis

We performed a preliminary analysis by assessing the radius of gyration ($R_g$) as an initial indicator of system equilibration. For all systems analyzed, the radius of gyration stabilized after approximately 200 ns (Fig. S8.A, B). While $R_g$ alone does not definitively prove system equilibration, it enables us to assess the convergence of the overall dimension of the nanodisc convergence point. We analyzed the

time evolution of peptide secondary structure over the full simulation duration of 6.8 µs at 30 °C and 37 °C. This analysis revealed that the $\alpha$-helical content of the peptides remained consistently stable, with no significant changes observed (Table. S2).

We calculated mass density profiles to determine nanodisc diameters and the positional relationship between peptides and lipid patches (Fig. S9). In all simulations, no peptides detached from their associated lipid patches. We report the density-derived average diameters of the nanodiscs, with their associated uncertainties, at 30 °C and 37 °C, and we do not observe any marked change in nanodisc diameter upon increasing the temperature (Table. S2).

## 3.2. Validation of 14A peptide model

We compared our simulation results with solid-state NMR (ssNMR) measurements [27], specifically examining the $^{15}$N chemical shifts of residues Ala5 and Leu10, and the $^2$H-NMR quadrupolar splittings of Ala5 and Ala13 in 14A peptides around lipid bilayer patch. These parameters are sensitive to the orientation of specific bonds: the C-N and N-H bonds for the $^{15}$N chemical shifts, and the $C_a-C_b$ bonds for the $^2$H quadrupolar splittings. The computational approach employed for calculating these NMR parameters is detailed in Section 2.4.

The average chemical shifts and quadrupolar splittings obtained from simulations of the 14A/DMPC nanodisc systems are summarized in Tables 2 and 3. The quadrupolar splittings ($^2$H) demonstrate consistency across nanodisc systems of varying sizes. Specifically, the calculated splittings for Ala5 consistently matches experimental data.

A notable discrepancy arises for Ala13. As a terminal residue, Ala13 is expected to experience enhanced motional freedom in the MD simulations, which likely leads to an overestimation of motional averaging and thus an underestimation of the time-averaged $^2$H splitting. In earlier solid-state NMR work [46], a $^2$H quadrupolar tensor derived from $^2$H-Ala-labeled peptide powder at room temperature (maximum splitting of 74 kHz) was used as a reference. This is a sensible starting point for probing fluctuations in fluid-phase bilayers. However, in MD we cannot distinguish the intrinsic peptide mobility (visible in the powder at room temperature) from the additional fluctuations associated with the fluid membrane phase. For this reason, we employed a full $^2$H quadrupolar tensor of 84 kHz for the MD back-calculations. This adjustment has only a minor quantitative impact and does not, by itself, reconcile the Ala13 splitting with the experiment.

As a complementary, we computed splittings directly from the mean angles of Ala5 and Ala13 using a prefactor of 74 kHz (i.e., neglecting membrane-induced motional averaging, as in the experiment [27]). Using the corresponding mean angles approximately 45° for Ala5 and 90° for Ala13, the resulting splittings are +18.5 kHz and -37 kHz, respectively. These values correspond directly to the experimentally observed magnitudes.

Experimentally, two points are important. First, the sign of the $^2$H splitting cannot be determined directly. Consequently, the splitting-versus-angle relationship (Fig. S10) admits multiple angle intervals that yield similar splitting magnitudes (Table 4) when evaluated with a 74 kHz prefactor, as in the experiment. Second, typical experimental uncertainties are < 4 kHz, which translates to angular uncertainties of < 5°.

By contrast, MD simulations provide the sign implicitly by back-calculating from the instantaneous geometries. While this is advantageous, MD-derived averages are subject to the larger thermal fluctuations and shorter accessible timescales relative to experiment. Taking these limitations into account, the well-established angular dependence of the $^2$H quadrupolar splitting and its high sensitivity to $\theta$ nonetheless indicate that the angles obtained from our trajectories are robust, and the corresponding back-calculated $^2$H splittings are fully consistent with the experimental data.

The back-calculated $^{15}$N chemical shifts of Ala5 and Leu10, based on the peptide plane orientations, closely match the experimental values.
<caption>**Table 2**</caption>

Comparison of theoretically obtained ²H quadrupolar splittings and ¹⁵N chemical shifts for Ala5, Ala13, and Leu10 in System A with ssNMR experimental data. Theoretical values were calculated from molecular dynamics simulations and absolute values are reported. The Global row represents values computed by concatenating all independent simulation runs into a single trajectory before calculating the ssNMR parameters, to capture the overall averaged behavior. Experimental ssNMR* values are taken from Salnikov et al. [27].

<table><caption>**Table 3**</caption><thead><tr><th rowspan="2">SYSTEM A</th><th colspan="2">²H-Ala5</th><th colspan="2">²H-Ala13</th><th rowspan="2">¹⁵N-Ala5 (ppm)</th><th rowspan="2">¹⁵N-Leu10 (ppm)</th><th rowspan="2">Temp (°C)</th><th rowspan="2">Time (ns)</th></tr><tr><th>Splitting (kHz)</th><th>Angles (°)</th><th>Splitting (kHz)</th><th>Angles (°)</th></tr></thead><tbody><tr><td>Run1</td><td>20 ± 26</td><td>46 ± 16</td><td>20 ± 18</td><td>90 ± 23</td><td>92 ± 27</td><td>76 ± 17</td><td>30</td><td>1000</td></tr><tr><td>Run2</td><td>17 ± 23</td><td>47 ± 14</td><td>21 ± 17</td><td>91 ± 23</td><td>91 ± 21</td><td>73 ± 16</td><td>30</td><td>1000</td></tr><tr><td>Run3</td><td>24 ± 24</td><td>43 ± 14</td><td>21 ± 16</td><td>89 ± 21</td><td>87 ± 24</td><td>80 ± 23</td><td>30</td><td>1000</td></tr><tr><td>Global</td><td>20 ± 25</td><td>45 ± 15</td><td>20 ± 18</td><td>90 ± 23</td><td>91 ± 25</td><td>76 ± 19</td><td>30</td><td>3000</td></tr><tr><td>Run1</td><td>15 ± 27</td><td>44 ± 14</td><td>18 ± 19</td><td>89 ± 25</td><td>96 ± 28</td><td>75 ± 16</td><td>37</td><td>1000</td></tr><tr><td>Run2</td><td>23 ± 24</td><td>49 ± 12</td><td>21 ± 16</td><td>89 ± 22</td><td>87 ± 22</td><td>75 ± 16</td><td>37</td><td>1000</td></tr><tr><td>Run3</td><td>13 ± 22</td><td>48 ± 16</td><td>18 ± 18</td><td>91 ± 25</td><td>97 ± 24</td><td>70 ± 13</td><td>37</td><td>1000</td></tr><tr><td>Global</td><td>17 ± 24</td><td>47 ± 14</td><td>19 ± 18</td><td>90 ± 24</td><td>93 ± 25</td><td>72 ± 16</td><td>37</td><td>3000</td></tr><tr><td>ssNMR*</td><td>19 ± 4</td><td>N/A</td><td>35 ± 2</td><td>N/A</td><td>96 ± 3</td><td>71 ± 2</td><td>37</td><td>N/A</td></tr></tbody></table>

Comparison of theoretically obtained ²H quadrupolar splittings and ¹⁵N chemical shifts for Ala5, Ala13, and Leu10 in System B with ssNMR experimental data. Theoretical values were calculated from molecular dynamics simulations and absolute values are reported. The Global row represents values computed by concatenating all independent simulation runs into a single trajectory before calculating the ssNMR parameters, to capture the overall averaged behavior. Experimental ssNMR* values are taken from Salnikov et al. [27].

<table><caption>**Table 4**</caption><thead><tr><th rowspan="2">SYSTEM B</th><th colspan="2">²H-Ala5</th><th colspan="2">²H-Ala13</th><th rowspan="2">¹⁵N-Ala5 (ppm)</th><th rowspan="2">¹⁵N-Leu10 (ppm)</th><th rowspan="2">Temp (°C)</th><th rowspan="2">Time (ns)</th></tr><tr><th>Splitting (kHz)</th><th>Angles (°)</th><th>Splitting (kHz)</th><th>Angles (°)</th></tr></thead><tbody><tr><td>Global</td><td>18 ± 23</td><td>46 ± 13</td><td>22 ± 16</td><td>89 ± 22</td><td>94 ± 23</td><td>72 ± 17</td><td>30</td><td>400</td></tr><tr><td>Global</td><td>17 ± 27</td><td>47 ± 16</td><td>19 ± 18</td><td>89 ± 24</td><td>95 ± 26</td><td>78 ± 22</td><td>37</td><td>400</td></tr><tr><td>ssNMR*</td><td>19 ± 4</td><td>N/A</td><td>35 ± 2</td><td>N/A</td><td>96 ± 3</td><td>71 ± 3</td><td>37</td><td>N/A</td></tr></tbody></table>

Experimental ²H splitting values for Ala5 and Ala13 and the corresponding possible angle intervals derived from the splitting-vs-angle plot. Because the experimental sign of the splitting cannot be directly determined, all angle intervals that yield positive or negative splittings consistent with the measured magnitude are reported. Note: The possible angle intervals (second column) are calculated in the static peptide case with no membrane fluctuations included.

<table><thead><tr><th>Residues</th><th>Possible angle intervals (°)</th><th>Exp.value (kHz)</th></tr></thead><tbody><tr><td>Ala5</td><td>(43 to 47), (64 to 79), (112 to 116), (133 to 137)</td><td>19.0 ± 4</td></tr><tr><td>Ala13</td><td>(35 to 38), (78 to 102), (143 to 147)</td><td>34.5 ± 2</td></tr></tbody></table>

In particular, mean chemical shift values of Ala5 and Leu10 below 100 ppm indicate that the labeled ¹⁵N-H vectors align approximately perpendicular to the membrane normal (Fig. 18 in [44], Fig. 4 in [48]), coinciding closely with the peptide helix axis. The standard deviations observed in chemical shift values reflect both the intrinsic dynamics of the peptide captured during simulations and the thermal fluctuations occurring throughout the simulation.

Despite these thermal fluctuations, the NMR parameters derived from our simulations remain in good agreement with experimentally observed data. It is worth noting that similarly, uncertainties (standard deviations) from mean values are commonly observed in comparative studies between molecular simulations and solid-state NMR experiments. [52–54]. Although the experiments were performed at 37 °C, we found no differences in the calculated NMR parameters between 30 °C and 37 °C. Hence, we conducted all subsequent analyses at 37 °C, unless otherwise stated. Therefore, our results strongly support the reliability of the 14A/DMPC models for further characterization.

### 3.3. Peptide-peptide interactions

Peptide-peptide interactions play a pivotal role in stabilizing nanodiscs. In contrast to MSP-based nanodiscs [19], APOA1-mimetic peptides rely exclusively on non-covalent interactions to maintain their

association with the discoidal lipid bilayer [23–25]. NMR studies on 14A peptides suggest an antiparallel arrangement of peptides forming dimers, which leads to a belt-like organization around the discoidal bilayer [27]. Guided by this proposed structural model, we set up and performed molecular dynamics simulations to replicate the peptide-lipid assembly. Theoretical back-calculated NMR parameters from the MD simulations closely agree with experimental data, supporting the validity of the antiparallel configuration and the orientation of the peptide axes nearly perpendicular to the nanodisc normal (Tables 2 and 3). These simulations further enabled a detailed characterization of the molecular interactions underlying this structural organization.

In particular, MD simulation highlights the role of central tyrosine residues (Y7) in aromatic π–π stacking interactions with adjacent peptides (Fig. 2A). The para-oriented hydroxyl groups of the tyrosines are positioned away from the interacting aromatic rings, favoring effective π–π stacking. This interaction is reflected in the free energy profile, which exhibits two distinct minima at 4.3–4.7 Å and the angle between the ring normal of 0°–10° and 170°–180°, corresponding to the stacked configurations where the aromatic ring plane remains parallel and aligned. The energy barrier separating the two minima is < 1 kcal/mol, so the rings can flip between two equivalent orientations without disrupting the stacking. The average intermolecular distance for these interactions is approximately 4.5 Å in both system sizes studied (Fig. 2B, D), indicating that Y7–Y7 stacking occurs near the interface region of the antiparallel peptides and contributes significantly to dimer stabilization.

Additionally, phenylalanine residues (F14), located at the peptide termini, participate in lateral interactions with F14 residues from adjacent dimer blocks (Fig. 2A). While these interactions contribute to the overall belt stabilization, they exhibit lower persistence in the bounded conformation compared to Y7-mediated π–π stacking. The two-dimensional free-energy maps for phenylalanine pairs (F14–F14) in smaller and larger nanodiscs (Fig. 2C, E) share the same fingerprint: a continuous, low-energy ridge that runs almost the full 0°–180° range of ring-normal angles but is tightly confined to the distance of ≈ 4–6 Å. Rather than settling into a few discrete wells, the rings populate a
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>**Fig. 2.** (A) Schematic depiction of peptide interactions illustrating both the formation of dimeric units and the lateral contacts established between adjacent dimers. (B) Free energy profile of tyrosine residues (Y7) located at the dimer interfaces for the system A, while (D) shows the corresponding profile for the system B. (C) Free energy profile of phenylalanine residues (F14), which mediate lateral interactions between neighboring dimer blocks in system A, while (E) shows the corresponding profile for the system B.</figcaption></figure>

continuum of orientations while maintaining a characteristic $π – π$ distance. The result is a lateral network that is cohesive yet highly flexible, precisely the mechanical property needed for the peptide belt to expand, contract, or ovalize, required to adapt to lipid fluctuations across nanodiscs of different diameters without losing structural integrity.

Together, these non-covalent interactions enable peptides to remain as stable dimers, while dynamic interactions between dimer blocks help maintain the peptide axes nearly perpendicular to the nanodisc normal. The peptides orient in such a way that their hydrophobic residues cover the lipid tails, creating a predominantly hydrophobic interface between the peptides, while polar residues effectively shield these hydrophobic surfaces (Fig. S12). Collectively, these findings support a conserved belt-like organization of peptides around the discoidal bilayer, observed consistently across all simulated systems (Table 1).

### 3.4. Lipid-peptide interactions

Having characterized the interactions among peptides surrounding the discoidal structure, we further explored their interactions with phospholipid molecules. Due to the amphipathic nature and specific arrangement of the peptides, we anticipated that polar and aromatic amino acids at the lipid-peptide interface would interact with both the DMPC head groups and the hydrophobic aliphatic tails.

Previous simulation studies have indicated the active involvement of polar amino acids at this interface in peptide-phospholipid interactions [55,56]. Consistent with these earlier findings, lysine (K9) and glutamic acid (E12) residues formed salt bridges with the DMPC head groups, adopting a “snorkel-like” orientation at the rim. Specifically, the amnio group ($NH_3^+$) of the lysine side chain interacts with the phosphate group of DMPC, and the carbonyl group of glutamic acid
<caption>**Table 5**</caption>

Interactions were quantified as the average number of contacts per peptide between: (i) the NH<sub>3</sub><sup>+</sup> group of Lys (K9) and the phosphate group of DMPC (salt bridge), (ii) the carboxylate of Glu (E12) and the choline group of DMPC (salt bridge), and (iii) the hydroxyl of Tyr (Y2) and the choline quaternary ammonium of DMPC (ion-dipole). Values are averaged over simulation frames and normalized by the number of peptides.

<table><thead><tr><th rowspan="2">System</th><th colspan="3">No. of Interactions per Peptide</th></tr><tr><th>Lys-Phos (salt bridge)</th><th>Glu-Choline (salt bridge)</th><th>Tyr-Choline (ion dipole)</th></tr></thead><tbody><tr><td>A</td><td>0.50</td><td>0.43</td><td>0.68</td></tr><tr><td>B</td><td>0.71</td><td>0.68</td><td>0.61</td></tr></tbody></table>

interacts with the choline group of DMPC (Table 5) (Fig. 3A). Despite the presence of three lysine residues per peptide, the peptide orientation allows only the lysine (K9) residue at the lipid-peptide interface to selectively engage with the phosphate groups of lipid head groups. Additionally, tyrosine (Y2) has a hydroxyl group at the para position of its aromatic ring, facilitating ion-dipole interactions with the choline group of the DMPC (Fig. 3B) (Table 5).

Furthermore, aromatic residues, such as phenylalanine (F6) and tyrosine (Y2), also interact significantly with the hydrophobic lipid tails. This interaction was quantified by calculating the angle between the normal of the aromatic ring plane and the membrane normal (z-axis). The resulting distribution showed that aromatic ring planes predominantly orient perpendicular to the membrane normal, effectively parallel to the lipid bilayer plane, thus acting as anchors (Fig. 3B, C) (Fig. S14). Collectively, these non-covalent interactions stabilize the peptide arrangement like a “belt”, and may influence the lipid properties near the nanodisc rim.

### 3.5. Lipid properties

Based on previous experimental studies [57,58] and simulation data [59,60], nanodisc lipid bilayers exhibit properties that deviate slightly from those of pure lipid bilayers. Nanodiscs incorporate membrane scaffold proteins (MSPs), and the interactions between lipids and proteins induce shape fluctuations that influence lipid properties. However, no simulation studies to date have examined nanodiscs composed specifically of DMPC lipids stabilized by apoA1 mimetic peptides. To address this gap, we investigated the lipid properties of a 14A/DMPC nanodisc model by analyzing the DMPC bilayer thickness and the order parameter ($S_{CH}$) of DMPC lipid chains at temperatures of 30 °C and 37 °C.

To facilitate analysis, the lipid area within the nanodiscs was divided into zones based on their distance from the peptides specifically, above or below 10 Å following established methods [59–61]. Our results reveal consistent trends in both bilayer thickness and lipid order parameters across temperatures and nanodisc sizes. Specifically, we compared the properties of 14A/DMPC nanodiscs to those of a pure DMPC bilayer slab and observed clear rim-to-core heterogeneity. In both small and large nanodiscs, lipids located near the rim adjacent to the apoA1-mimetic peptide belt were significantly thinner and less ordered, while the core lipids were more ordered and tightly packed.

In the small nanodisc (System A), the rim-to-core inhomogeneity is more pronounced: lipids at the center are actually more ordered than in a pure DMPC bilayer, whereas those within ≈10 Å of the peptide belt are thinner and highly disordered (Fig. 4A). A rise in temperature from 30 °C to 37 °C lowers both the average bilayer thickness and the order parameter across the disc, but the core remains distinctly tightly packed than the rim. In the larger nanodisc (System B) we observed the same trend but it is less pronounced. At 30 °C, the nanodisc core is almost similar to a pure DMPC bilayer; only a rim region is noticeably thinner and more disordered (Fig. 4C). Raising the temperature to 37 °C

induces uniform thinning and a modest drop in chain order at both rim and core (Fig. 4D). Even so, the core still exceeds the ordering seen in a pure DMPC bilayer, while the rim remains the most perturbed region.

Both system A and system B showed a slight decrease in thickness and lipid order near the rim and core as temperature increased, but the overall diameter remains the same (Fig. 4A-D; Table. S2). This temperature-induced decrease in thickness and lipid order is consistent with previous findings in MSP1/DMPC-based nanodiscs at temperatures above the phase transition of DMPC (Fig. 7 in [62]). In nanodiscs of different sizes and at both temperatures we studied, the lipids in the core are always thicker and more ordered than in a pure DMPC bilayer.

These findings align well with previous simulation studies on DMPC nanodiscs involving various MSP constructs, including MSP1, MSP1E1, MSP1E2 [59,60,62] and (Δ)-H5 proteins [57,58,61].

### 4. Conclusion

We conducted a detailed investigation of the structural and dynamic properties of lipid nanodiscs assembled with the apoA1 mimetic peptide 14A, utilizing all-atom molecular dynamics simulations. Peptide orientation derived from MD simulations was validated by comparing the ²H-NMR quadrupolar splittings and ¹⁵N chemical shifts of specific residues with solid-state NMR data, yielding good agreement.

We thoroughly analyzed the dimeric arrangement of the 14A peptide, addressing speculations [27] raised in earlier studies and characterizing the key interactions involving in stabilizing the dimers. Notably, aromatic $π-π$ interactions between tyrosine residues (Y7) in antiparallel oriented peptides stabilize the dimeric arrangement. Phenylalanine (F14) residues positioned at the interfaces between neighboring dimer blocks form $π-π$ contacts that are less persistent than the dimer-stabilizing tyrosine (Y7) interactions. Their transient nature can lead to small cleavages at these interfaces. Although our simulations did not explicitly capture lipid exchange, experimental studies demonstrate that peptide-based nanodiscs exchange lipids during particle collisions via such cleavages, providing a low-energy pathway for lipid exchange [63, 64]. Unlike nascent apoA1 nanodiscs, which exchange lipids primarily through diffusion, as observed in time-resolved small-angle neutron scattering (SANS) measurements in aqueous solution [65].

Furthermore, we characterized specific lipid-peptide interactions. Polar amino acids, including lysine (K9) and glutamic acid (E12), engage with the charged headgroups of DMPC lipids through electrostatic interactions. Aromatic residues, particularly tyrosine (Y2) and phenylalanine (F6), anchor peptides consistently to the hydrophobic lipid tails. Notably, the hydroxyl group of tyrosine (Y2), positioned para to its aromatic ring, interacts specifically with the choline groups of DMPC lipids. We compared lipid properties of 14A/DMPC nanodiscs against a pure DMPC bilayer slab, revealing clear rim-to-core heterogeneity. In both nanodisc sizes, lipids at the rim near the apoA1-mimetic peptide belt were markedly thinner and less ordered. Temperature-dependent thinning and decreased lipid order near the rim and core were similarly observed in both sizes. Based on earlier studies of
<figure><img src="image_8.png" /><figcaption>**Fig. 3.** Representation of system A configuration sampled during molecular dynamics (MD) simulation, highlighting key interactions and orientations. (A) Salt bridge interactions between peptides and lipid head groups: a lysine (K9) - phosphate salt bridge formed by interactions between the amino group (NH₃⁺) of the lysine (blue) and the phosphate group of DMPC (orange), and a glutamic acid (E12) - choline salt bridge formed by interactions between the glutamate carboxyl group (red) and choline group of DMPC (yellow). (B) Orientation of aromatic residues along the bilayer normal, with tyrosine (green) and phenylalanine (red) rings aligned nearly parallel to the membrane normal. Additionally, an ion-dipole interaction is observed between the hydroxyl group (red ball) in the para position of the tyrosine aromatic ring and the choline group of the DMPC (yellow). (C) Probability distribution of the aromatic ring normal orientations for tyrosine and phenylalanine within the 0°–180° range of system A.</figcaption></figure>

MSP-based nanodiscs [23,61,62] and our results, we can speculate that the direct lipid-peptide interactions can induce additional disorder to the lipid tails near the rim. Over, our results are consistent with earlier findings on MSP/DMPC-based nanodiscs [23,57,59,60] [58,61] suggest similar lipid properties as MSP-based nanodiscs could be expected in the peptide-based nanodiscs.

The short length of the 14A peptide makes it easily synthesiz-
able, yet it effectively retains interactions necessary for stabilizing
lipid bilayer patches, closely mimics lipid properties of the MSP-based
nanodiscs, and demonstrates an ordered dimeric arrangement of pep-
tides in the apoA1 mimetic peptide family. These 14A peptide-based
nanodiscs maintain efficient alignment capabilities in magnetic fields
suitable for solid-state NMR studies and offer size tunability [23]. Thus,

they serve as promising platforms for detergent-free membrane protein
characterization. Moreover, the ability of 14A peptides to stabilize
lipid bilayer patches via strong lipid-peptide interactions could further
facilitate the study and development of active therapeutic agents for
cardiovascular disease.

In summary, our atomistic MD simulations provide a coherent
structural and dynamic description of 14A/DMPC nanodiscs that is con-
sistent with available ssNMR data [27]. The results provide evidence in
support of the hypothesis that 14A anti-parallel dimers organize into a
belt-like arrangement around the lipid bilayer patch [27] and reveal the
key peptide-peptide and peptide-lipid interactions that stabilize this
scaffold. Beyond rationalizing existing experiments, our study provides
a robust foundation for further investigations and paves the way for
<figure><img src="image_9.png" /><figcaption>**Fig. 4.** 2D maps of bilayer thickness (left panels), average lipid order parameters calculated over all C-H bonds in the two aliphatic tails of DMPC lipids (center panels), and order parameters as a function of carbon position along the lipid tails (right panels). The rim zone is defined as all lipids within 10 Å of the 14Å peptides, while the core zone includes all lipids located beyond this distance. Panels (A) and (B) correspond to System A, simulated at 30 °C and 37 °C, respectively. Panels (C) and (D) correspond to System B, simulated at 30 °C and 37 °C, respectively.</figcaption></figure>

similar future studies, such as those involving nanodiscs formed by the 18Å peptides [66] and other apoA-I mimetic sequences [26].

### CRediT authorship contribution statement

**Rohith Ravi:** Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Evgeniy S. Salnikov:** Writing – review & editing, Visualization, Validation, Methodology, Formal analysis. **Burkhard Bechinger:** Writing – review & editing, Supervision, Resources, Funding acquisition. **Mounir Tarek:** Writing – review & editing, Supervision, Conceptualization.

### Funding

The financial contributions of the Agence Nationale de la Recherche (projects AmphiPep 20-CE18-0021, SAFEST 21-CE18-0043, AMLAA 21-CE11-0038-02, the Interdisciplinary Thematic Institute SysChem, via the IdEx Unistra (ANR-10-IDEX-0002) within the French Investments for the Future Program), the CAPES-COFECUB (88881.879071/2023-01) program for international exchange (CAMPUS France).

### Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
MOUNIR TAREK reports financial support was provided by French National Research Agency. BURKHARD BECHINGER reports financial support was provided by French Investments for the Future Program. BURKHARD BECHINGER reports financial support was provided by CAPES-COFECUB. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Appendix A. Supplementary data

Supplementary material related to this article can be found online at https://doi.org/10.1016/j.bbamem.2025.184495.

## Data availability

Data will be made available on request.

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