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Complexation of peptide epitopes with G4-PAMAM dendrimer through ligand diffusion molecular dynamic simulations
Journal of Molecular Graphics and Modelling 96 (2020) 107514
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Topical Perspectives
Complexation of peptide epitopes with G4-PAMAM dendrimer through ligand diffusion molecular dynamic simulations Correa-Basurto Martiniano Bello*, Rolando Alberto Rodríguez-Fonseca, Jose rmacos y Bioinforma tica, Escuela Superior de Medicina, Instituto Polit ~ o de fa Laboratorio de Modelado Molecular, Disen ecnico Nacional, Plan de San Luis y n, Ciudad de M Díaz Miro exico, 11340, Mexico
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 September 2019 Received in revised form 12 December 2019 Accepted 16 December 2019 Available online 20 December 2019
Peptide epitopes from HIV-1 gp120 have been used to block the gp120-CD4 complex, whereas their poor absorbable or immunogenic properties prevent them from coupling to generation four polyamidoamine (PAMAM-G4) dendrimers. PAMAM-G4 are synthetic nanoparticles that are relatively nontoxic and nonimmunogenic have been employed as nanocarriers. In a previous study, two peptide epitopes (ABC and PGV04) from gp120 located at the protein-protein interface of the gp120-CD4 complex were identified through protein-protein dissociation. Then, their complexation with G4-PAMAM was evaluated through experimental and theoretical approaches, revealing a stoichiometry of 1:8/9 for G4-PAMAM and ABC or PGV04, respectively, providing important information that can be used to gain insight into the structural and energetic basis of the molecular binding of these G4-PAMAM-peptide systems. In this contribution, we performed ligand diffusion molecular dynamic simulations (LDMDSs) using 1.5 ms combined with the molecular mechanics generalized Born surface area (MMGBSA) approach, a strategy that successfully reproduced experimentally encapsulation on PAMAM-G4-ligand complexes, to explore the mechanism through which ABC and PGV04 are encapsulated by PAMAM-G4 under neutral and acid conditions. Our results reproduce the reported PAMAM-G4-peptide complex stoichiometry, revealing a slower peptide delivery at neutral conditions and a spontaneous release under acidic conditions. LDMDSs show that several peptides can reach stable G4-PAMAM complexes at neutral pH, and only a few are able to encapsulate on dendrimers without impacting dendrimer sphericity. Energetic analysis exploring different generalized Born models revealed that the ABC peptide has better binding properties than PGV04. © 2019 Elsevier Inc. All rights reserved.
Keywords: PAMAM-G4 Dendrimer Peptide Ligand diffusion molecular dynamics simulations Binding free energy
1. Introduction The protein gp120 is the first HIV-1 glycoprotein that establishes contact with host target cells on the CD4 receptor [1]. Blocking gp120-CD4 complex formation may prevent HIV-1 infection in host CD4þ target cells [2]. Thus, three-dimensional (3D) models of the quaternary structure of HIV-1 envelope proteins (gp120-gp41) forming complexes with broadly neutralizing antibodies (bnAbs) from cryo-electron microscopy (PDB: 3J5M) are valuable resources for peptide epitopes. This gp120-gp41 complex displays a wellordered quaternary structure that permits a good antigenic profile and as native gp120-gp41 trimers, discriminates between recognition by bnAbs and non-bnAbs [3]. Thus, this structural
* Corresponding author. E-mail address: [email protected] (M. Bello). https://doi.org/10.1016/j.jmgm.2019.107514 1093-3263/© 2019 Elsevier Inc. All rights reserved.
information could be exploited to develop HIV-synthetic peptide epitope vaccines that are safer, cheaper and immune-targeted to CD4 binding sites, a vulnerability domain of HIV-1 [4]. However, challenges faced by peptide epitopes, such as instability in the presence of endogenous enzymes, poor immunogenicity and poor antigen-presenting cell (APC) uptake, limit the development of HIV-synthetic peptide epitope vaccines. These challenges could be resolved by using dendrimer-based delivery platforms. Dendrimers are branched nanoparticles used as drug-delivery approaches with high water solubility [5] and biocompatibility [6]. Dendrimers can encapsulate ligand molecules by nonbonding interactions inside their empty cavities present around the core [7]. In addition, these dendrimer platforms have previously been demonstrated to enhance the peptide-specific immune response by acting as adjuvants [8,9], enhancing antigen uptake by APCs [10] and improving the vaginal-immune response of synthetic-peptide vaccines [8,9]. However, prior to evaluating the capability of dendrimers to
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improve ligand properties of the peptide, the dendrimer-peptide complexes need to be characterized by physicochemical procedures [8]. Theoretical procedures are valuable tools for screening peptide ligands that are able to form dendrimer complexes. However, in silico characterization methods for dendrimer-ligand complexes involve small molecule ligands, and traditional methods of molecular docking have limitations mainly for aqueous environment simulations [11], which is because these methods do not consider solvation energies and algorithm limitations in peptide molecules with higher rotatable bonds or higher conformational-space [11]. Due to this limitation, docking and MD simulations have been combined to get insight into the energetic basis of the molecular recognition and encapsulation of small ligands, peptides and nucleic acids by native or modified dendrimers [13e18]. In this context, Marquez-Miranda et al. combined docking and MD simulations to evaluate the ability of G4-PAMAM-OH dendrimers to bind angiotensin peptides of seven amino acids, revealing that this dendrimer is able to bind three angiotensin peptides through docking calculations, whereas MD simulations showed that the dendrimer-peptide complex was stabilized through electrostatic and vdW interactions [12]. However, a drawback of this latter study may be the restriction of rotatable bonds for peptides higher than 7 amino acids present in the AutoDock 4.2 suite. Ligand diffusion inside proteins have been explored through MD simulations in combination with several methodological approaches that apply forces to the ligand to accelerate the binding or unbinding process [19e21]. Recently, a theoretical method named ligand diffusion molecular dynamic simulations (LDMDSs) combined with the molecular mechanics generalized surface area (MMGBSA) approach was employed to explore the mechanism through which methotrexate (MTX) is complexed to G4-PAMAM dendrimers at neutral, basic, and acidic conditions. The theoretical approach was able to reproduce experimental stoichiometry between MTX and PAMAM-G4 under neutral conditions, whereas the binding free energy value suggested a much slower release of MTX in neutral conditions compared to acidic conditions, and poor affinity was observed under basic conditions [22]. Under the LDMD approach, one of several ligands is positioned at different distances from the receptor, permitting molecular recognition through conventional MD simulations without employing any external bias that forces binding. This approach has been used to explore the complex formation between proteins and small organic molecules [23e25], whereas LDMDs in combination with the MMGBSA approach have been used to observe ligand diffusion on proteins [26] and on PAMAM-G4 dendrimers [22]. However, as far as we are concerned, this method has not been employed to assess the loading and encapsulation properties of peptides on the PAMAM-G4 dendrimer. In a previous study, experimental and theoretical approaches, including docking studies followed by MD simulations, were employed to elucidate the binding stoichiometry of two peptide epitopes (ABC and PGV04) identified from gp120 after structural dissection of the gp120-CD4 complex [27], revealing a stoichiometry of 1:8 and 1:9 for G4-PAMAM and the ABC or PGV04 peptides, respectively, providing important information about the structural complexation but lacking important structural and energetic information about the affinity and encapsulation properties of both peptides by G4-PAMAM dendrimers under neutral and acidic conditions, which represent the physiologic conditions of vaccine transportation and delivery, respectively. Therefore, this represents a good system to evaluate the ability of LDMDSs combined with the MMGBSA approach to reproduce experimental stoichiometry and provide structural and energetic information that is difficult to obtain through experimental approaches and docking studies. In
this study, we employed LDMDs together with the MMGBSA approach in the microsecond (ms) time scale using saturating concentrations of the ABC and PGV04 peptides to explore the mechanism through which these peptides are complexed with PAMAMG4 at neutral and acidic conditions to make information concerning the energetic and encapsulation properties available. 2. Material and methods 2.1. Ligand diffusion MD simulations The three-dimensional structures of the PGV04 (SFANSSGGDLEV) and ABC (GSTNSTTETFRPGGGD) peptides were taken from a previous study [8]. Each peptide conformation was placed around the charge-neutral and charge-acid G4-PAMAM at a peptide ligand to G4-PAMAM ratio of 10:1 at different distances that oscillated between 7.0 and 15 Å from the G4-PAMAM using AmberTools 16 [28]. Force field parameters and tridimensional data for chargeneutral PAMAM-G4 were taken from the website http://www. physics.iisc.ernet.in/~maiti/dbt/home.html, and the force field parameters and tridimensional data for charge-acid PAMAM-G4 were taken from a previous work [13]. LDMDSs were performed with the Amber 16 package together with the ff14SB force field [29] and the generalized Amber force field (GAFF) [30]. The topologies of the G4PAMAM-peptide complex systems were built by the LEaP module and minimized and equilibrated through the Sander module and pmemd.cuda Amber 16 [28] through the use of Graphical Units Processors [31]. Aspartic and glutamic acid amino acids of the PGV04 and ABC peptides were modeled as protonated and unprotonated for simulations under acid and neutral pH, respectively. Chlorine atoms were placed at different locations by LEaP to mimic physiological conditions and neutrality. A rectangular-shaped box of water was constructed using the TIP3P water model [32]; the water model was extended 20.0 Å between the G4-PAMAM and the limit of the box. Afterwards, the solvated and neutralized G4PAMAM was minimized and equilibrated by the following steps: minimization through 5000 steps of steepest descent minimization; 1000 picoseconds (ps) of heating; 1000 ps of density equilibration with weak restraints on the complex; and 10 ns of constant pressure equilibration at 310 K using the SHAKE algorithm [33] on hydrogen atoms to allow a time step of 2 femtoseconds (fs) and Langevin dynamics for temperature control. The equilibration run was followed by 250-ns-long MD simulations, without position restraints under periodic boundary conditions (PBC) and under an NPT ensemble at 310 K. The particle mesh Ewald method (PME) [34] was used to treat long-range electrostatic interactions under periodic conditions with a direct space cutoff of 10 Å, and a similar cutoff was employed for Van der Waals interactions. The time step of the MD simulations was set to 2.0 fs, and the SHAKE algorithm [33] was used to constrain bond lengths at their equilibrium values. Temperature and pressure were maintained using the weakcoupling algorithm [35] with coupling constants tT and tP of 1.0 and 0.2 ps, respectively (310 K, 1 atm). For each system, three independent 250-ns-long MD simulations with equal parameters were carried out. Coordinates were retrieved every 20 ps for analysis. AmberTools 16 was used to analyze the time-dependence of the solvent accessible surface area (SASA) and radius of gyration (RG) to obtain different dendrimer conformers. 2.2. Calculation of binding free energies Binding free energies of G4-PAMAM-peptide complexes were calculated using the MM/GBSA approach [36e38] provided in the Amber 16 suite. A total of 1000 snapshots were selected at each 100 ps time interval from the equilibrated simulation time for G4-
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PAMAM-peptide complexes (150-ns-long simulations). The solvation free energy (DGsol) was determined with three different generalized Born (GB) continuum solvation models implemented in Amber16: the pairwise GB model of Hawkins and colleagues (GB1) [39e41], and two modified GB models developed by Onufriev and collaborators (GB2 and GB5) [42]. The relative binding free energy (DGmmgbsa) of each G4-PAMAM-peptide complex represented the average value of three independent simulations and was determined as described previously [13].
3. Results and discussion 3.1. Equilibrium of G4-PAMAM-peptide complexes Analysis of the 250-ns-long LDMDSs between peptides and charge-acid and charge-neutral G4-PAMAM (Fig. 1) shows that the charge-acid G4-PAMAM-ABC-peptide and charge-neutral G4PAMAM-PGV04-peptide equilibrated between 10 and 25 ns with average Rg and SASA values of 24.6 ± 0.40 and 21709 ± 306.70 Å, respectively, for the G4-PAMAM-ABC-peptide system and 24.8 ± 0.37 and 21658 ± 338 Å, respectively, for the G4-PAMAMPGV04-peptide. G4-PAMAM-ABC-peptide and G4-PAMAM-PGV04peptide equilibrated between 25 and 50 ns with average Rg and SASA values of 21.14 ± 0.60 and 14035.95 ± 699.50 Å, respectively, for the G4-PAMAM-ABC-peptide system and 20.73 ± 0.44 and 12843.46 ± 605.27 Å, respectively, for the G4-PAMAM-PGV04peptide. The Rg and SASA values were in line with those previously reported [13,22,43e49]. Comparison of the Rg and SASA values of G4-PAMAM at acidic and neutral pH with those of free G4PAMAM indicates that the coupling of peptides does not significantly impact the sphericity shape of G4-PAMAM, which is in contrast to previous results in which the saturation of the G4PAMAM dendrimer with methotrexate affected the shape of PAMAM-G4 with respect to its free state at neutral conditions [22].
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3.2. Dynamics of the complex formation The G4-PAMAM dendrimer comprises surfaces similar to pockets, where the walls and bottom of the pockets are apolar and the spikes form the edges of the pocket and are polar. At neutral pH, the primary amines are positively charged, which results in an electrostatic repulsion at the dendrimer surface, allowing the formation of a number of cavities [13]. The number of cavities is increased in acidic conditions as a result of the electrostatic repulsion between the positively charged primary and tertiary amines at the dendrimer surface and internal branches, allowing the formation of more cavities that exhibit better encapsulation properties for small organic molecules [22]. LDMDSs at 250 ns showed that from the ten ABC peptide (Fig. 2A) and PGV04 peptide (Fig. 2E) conformers around chargeneutral G4-PAMAM dendrimers, nine ABC peptides (Fig. 2B) and ten PGV04 peptides (Fig. 2F) conformations were able to form complexes with G4-PAMAM dendrimers at 50 ns. Ten ABC (Fig. 2C) and PGV04 (Fig. 2G) peptides reached stabilized G4-PAMAM-peptide complexes from 150 ns until the end of the MD simulation. On the other hand, 250 ns-long LDMDSs for the ABC and PGV04 peptides with the charge-acid G4-PAMAM showed that both peptides were unable to reach a stable conformation with G4-PAMAM (Fig. 3). Although structural analysis of the peptides coupled to neutral G4-PAMAM revealed differences in the degree of encapsulation of peptides by G4-PAMAM dendrimers (Fig. 2C and 2G) and the lack of affinity of both peptides with acid G4-PAMAM, more accurate structural analysis needs to be performed to evaluate the degree of encapsulation of peptides at neutral pH. Therefore, quantitative persistence of the G4-PAMAM-peptide complexes was evaluated by quantifying the distance between the center of mass (COM) of the ten ABC and PGV04 peptide conformations and the COM of tertiary amines from G4-PAMAM. As shown in Table 1, only conformer1, conformer2 and conformer5 of the ABC-peptides showed COM distances lower than the Rg value (21.08 ± 0.65 Å) of the free charge-neutral G4-PAMAM
Fig. 1. Radius of gyration and solvent-accessible surface area analysis of the charge-acid and charge-neutral G4-PAMAM-peptide complexes through 250 ns LDMDS together with the MMGBSA approach.
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Fig. 2. Molecular diffusion of ABC and PGV04 peptides on charge-neutral G4-PAMAM. Initial coordinates for the ABC (A) or PGV04 peptide (E) with charge-neutral G4-PAMAM dendrimers. Coordinate taken during the 50 ns-long MD simulation for the ABC (B) and PGV04 peptides (F) with charge-neutral G4-PAMAM dendrimers. Snapshot taken during the 250 ns-long MD simulation for the ABC (C) or PGV04 peptide (G) with charge-neutral G4-PAMAM dendrimers. Coordinate showing the three ABC (D) and PGV04 (H) peptides that encapsulate the G4-PAMAM dendrimer during the 250-ns-long MD simulation. The ABC and PGV04 peptides are shown in yellow spheres and G4-PAMAM dendrimers are shown in cyan spheres (nitrogen and oxygen atoms of peptides and dendrimers are depicted in blue and red color, respectively). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article).
Fig. 3. Molecular diffusion of the ABC and PGV04 peptides on charge-acid G4-PAMAM. Initial coordinates for the ABC (A) or PGV04 peptide (D) with charge-acid G4-PAMAM dendrimers. Coordinate taken during the 50 ns-long MD simulation for the ABC (B) and PGV04 peptides (E) with charge-acid G4-PAMAM dendrimers. Coordinate taken during the 250 ns-long MD simulation for the ABC (C) or PGV04 peptide (F) with charge-acid G4-PAMAM dendrimers.
dendrimer (Figs. 4 and 2D), suggesting that only these ABC-peptide conformers exhibit encapsulation and strong coupling, whereas the remaining ABC-peptide conformers showed superficial and weak coupling. Similarly, for PGV04, only three peptides were encapsulated on the G4-PAMAM dendrimer; conformer1, conformer3 and
conformer7 of the PGV04-peptides (Figs. 4 and 2H) displayed COM distances minor to the Rg value of the free charge-neutral G4PAMAM, indicating encapsulation and strong binding for these three peptide conformers, whereas the remaining PGV04-peptide conformers suggested superficial and low affinity to G4-PAMAM.
M. Bello et al. / Journal of Molecular Graphics and Modelling 96 (2020) 107514 Table 1 Distances between the center of mass (COM) of ABC or PGV04 peptide conformers and the COM of tertiary amines of G4-PAMAM. System ABC peptide Conformer 1 Conformer 2 Conformer 3 Conformer 4 Conformer 5 Conformer 6 Conformer 7 Conformer 8 Conformer 9 Conformer 10
COM 15.98 16.94 28.77 34.23 32.63 30.40 11.57 24.44 30.80 24.44
± ± ± ± ± ± ± ± ± ±
1.77 1.13 3.53 1.63 3.81 1.98 1.05 1.62 3.03 1.59
System PGV04 peptide Conformer 1 Conformer 2 Conformer 3 Conformer 4 Conformer 5 Conformer 6 Conformer 7 Conformer 8 Conformer 9 Conformer 10
COM 20.28 ± 3.04 23.84 ± 3.58 12.305 ± 1.10 22.19 ± 1.89 12.17 ± 0.90 28.53 ± 3.61 21.18 ± 2.21 12.86 ± 1.22 25.60 ± 1.07 29.27 ± 2.49
5
mer2 > conformer3 > conformer9 > conformer5 > con former4 > conformer6. Comparison of the ranking affinity employing the three GB solvation models revealed that despite the differences observed for conformer7 and conformer10 between the IGB2/IGB1 and IGB5 models, a similar tendency was observed for the coupling of the ABC peptides on the G4-PAMAM dendrimer. The ten PGV04 peptide conformers showed the following affinity ranking for the IGB1 model: conformer5 > confor mer4 > conformer7 > conformer8 > con former10 > conformer1 > conformer2 > conformer6 > con former9 > conformer3. For the IGB2 model, we identified the following affinity order: conformer5 > conformer4 > con former7 > conformer10 > conformer8 > conformer1 > conformer
Fig. 4. Center of mass (COM) of the ABC or PGV04 peptide with G4-PAMAM. Distances between the COM of ABC or PGV04 peptide conformers and the COM of tertiary amines of charge-neutral G4-PAMAM during the 250-ns-long LDMDSs.
Overall, this analysis reveals that despite the structural differences of both peptides, only three peptide conformers are able to be encapsulated by G4-PAMAM at neutral pH conditions. 3.3. Binding free energy Although structural analysis of the peptide encapsulation by G4PAMAM provides important information about the ability of small organic molecules or peptides to be transported by G4-PAMAM, it is also important to quantify the affinity of the peptides coupled to G4-PAMAM. Tables IeIII (supplementary material) show that nonpolar interactions mostly guided the molecular recognition between ABC or PGV04 and G4-PAMAM when the GB2 and GB5 models were employed, whereas the GB1 model showed that the relative binding free energy employing the MMGBSA approach (DGmmgbsa) was the consequence of the contribution of polar and nonpolar contributions which contributed mostly to G4-PAMAMpeptide complex stabilization. Comparison of the DGmmgbsa values between the ABC or PGV04 conformers and G4-PAMAM (Table 2) showed that the peptides were coupled to the G4-PAMAM surface with different affinities. The DGmmgbsa values between the ten ABC conformers with G4PAMAM exhibited the following affinity ranking when the IGB1 and IGB2 models were used: conformer1 > confor mer10 > conformer7 > conformer8 > conformer2 > conformer3 > conformer9 > conformer5 > conformer4 > conformer6. The IGB5 model showed the following rankings: conformer1 > conformer7 > conformer10 > conformer8 > confor
Table 2 Binding free energy components (kcal/mol) of G4-PAMAM-peptide complexes using the MMGBSA approach and three different continuum solvation models: GB1, GB2 and GB5. System GB1 G4-PAMAM-peptide complex ABC Conformer 1 90.38 ABC Conformer 2 45.73 ABC Conformer 3 35.89 ABC Conformer 4 30.03 ABC Conformer 5 31.47 ABC Conformer 6 11.31 ABC Conformer 7 61.76 ABC Conformer 8 50.64 ABC Conformer 9 34.53 ABC Conformer 10 65.53 PGV04 Conformer 1 47.96 PGV04 Conformer 2 40.88 PGV04 Conformer 3 37.67 PGV04 Conformer 4 64.06 PGV04 Conformer 5 98.51 PGV04 Conformer 6 40.24 PGV04 Conformer 7 58.52 PGV04 Conformer 8 58.06 PGV04 Conformer 9 38.22 PGV04 Conformer 10 54.64
(0.42) (0.43) (0.42) (0.27) (0.30) (0.24) (0.35) (0.43) (0.33) (0.41) (0.60) (0.38) (0.28) (0.36) (0.49) (0.48) (0.37) (0.31) (0.37) (0.52)
GB2
GB5
70.24 (0.35) 28.60 (0.34) 24.72 (0.34) 17.39 (0.21) 19.31 (0.22) 5.32 (0.16) 47.50 (0.31) 32.35 (0.30) 22.65 (0.24) 47.99 (0.33) 33.42 (0.45) 26.20 (0.28) 22.09 (0.23) 46.76 (0.23) 78.32 (0.43) 28.56 (0.48) 42.57 (0.30) 38.60 (0.28) 25.23 (0.29) 41.39 (0.42)
73.02 (0.38) 30.01 (0.37) 24.65 (0.37) 15.61 (0.23) 19.23 (0.24) 4.58 (0.17) 51.35 (0.35) 32.03 (0.31) 22.50 (0.26) 48.73 (0.41) 33.58 (0.47) 25.31 (0.30) 21.22 (0.28) 47.26 (0.31) 88.83 (0.49) 28.77 (0.50) 43.91 (0.33) 42.06 (0.31) 25.54 (0.32) 42.65 (0.45)
6 > conformer2 > conformer9 > conformer3. For IGB5, the following tendency was observed: conformer5 > confor mer4 > conformer7 > conformer10 > conformer 8 > conformer1 > conformer6 > conformer9 > con
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Fig. 5. Representative G4-PAMAM-peptide conformations at neutral pH. The three G4-PAMAM-ABC-peptide conformations: conformer1 (A), conformer2 (B) and conformer5 (C). The three G4-PAMAM-PGV04-peptide conformations: conformer 1(D), conformer3 (E) and conformer5 (F) that encapsulate in the stabilized complex.
former2 > conformer3. Comparative analysis of the ranking affinity employing the three GB solvation models revealed that the IG2 and IGB5 models exhibited a more similar ranking affinity among them than with IGB2; however, the three GB solvation models were able to identify the first four peptide conformers with the highest DGmmgbsa values (conformer5, conformer4, conformer7 and conformer8/10) and were able to identify the peptide conformer coupled with the less favorable DGmmgbsa value (conformer3). Estimation of the performance of the three IGB models revealed that although the IGB2 and IGB5 models were better at reproducing similar affinity tendencies for the coupling of peptides with PAMAM-G4, the IGB1 model was more suitable since it allows the estimation of the polar and nonpolar contributors to the DGmmgbsa
value, whereas the IGB2 and IGB5 models underestimate the polar contribution. Interestingly, this result indicated that the IGB1 model was more suitable for estimating binding affinities in the PAMAM-G4-peptide/protein system, which is in contrast to previous studies for complexes between PAMAM-G4 and small organic molecules [13] and protein-ligand systems [50], where the GB2 model showed better accuracy in describing the polar and nonpolar components than GB1 and GB5. Based on the DGmmgbsa values obtained with the IGB1 model and the number of peptides coupled to G4-PAMAM, PGV04 exhibited a more favorable binding affinity for G4-PAMAM than ABC (Table 2). Comparison of the DGmmgbsa values with respect to the distances between the COM of ABC or PGV04 peptide conformers and the
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COM of tertiary amines of G4-PAMAM (Table 1) showed that there was a relationship between the highest DGmmgbsa values observed between ABC conformation (conformer1, conformer2 and conformer7) with G4-PAMAM and low COM values. Similarly, a relationship between low COM values and highest DGmmgbsa values was observed between two PGV04 peptide conformations (conformer5 and conformer8) with G4-PAMAM. Interestingly, there was a relationship between the highest DGmmgbsa values and low COM values for some peptides but not others, as for the case of conformer3 of PGV04 (Tables 1 and 2), indicating that for the complexation of some peptides with G4-PAMAM, the peptide does not necessarily need to be encapsulated to show energetically favorable DGmmgbsa values, which is in contrast with the complexation between single small organic molecules and PAMAM-G4, where a correlation was observed between the degree of encapsulation and the binding affinity [13], but in line with LDMDS studies where G4-PAMAM was saturated with 20e30 methotrexate ligands at three different pH values [22].
charged peptides with G4-PAMAM indicates that the ABC peptide is a better candidate for use as an epitope peptide since it exhibits a lower affinity for G4-PAMAM than PGV04, allowing for easier release from the dendrimer. In addition, the in silico methodology employed in this study could be employed to reduce the cost of screening synthetic peptides capable of attaching, with stability, to G4-PAMAM to carry out experimental assays.
3.4. Structural analysis of complex formation
Appendix A. Supplementary data
Analysis of the interactions present in the three G4-PAMAMABC/PGV04-peptide and G4-PAMAM-PGV04-peptide complexes showed that both peptides were stabilized by polar interactions between polar atoms of polar and charged residues of ABC peptides with the polar primary and tertiary amines and amide groups of the PAMAM-G4 dendrimer (Fig. 5AeC). For PGV04, similar polar interactions occurred between polar, charged and hydrophobic residues with PAMAM-G4 cavities (Fig. 5DeF). Based on the participation of Asp7 and Glu9 of the ABC peptide and Asp9 and Glu11 of the PGV04 peptide in hydrogen bonding, it can be suggested that the protonated state modeled for these residues in LDMDs under acidic conditions (see methods) prevented peptide coupling on the charge-acid G4-PAMAM. However, the nonpolar interactions were the most important to stabilize the G4-PAMAMpeptide complex system, occurring between the nonpolar atoms of the peptides and the nonpolar atoms of the PAMAM-G4 dendrimer.
Supplementary data to this article can be found online at https://doi.org/10.1016/j.jmgm.2019.107514.
4. Conclusions In this study, we evaluated the ability of LDMDSs combined with the MMGBSA approach to reproduce the experimentally observed stoichiometry between the ABC and PGV04 peptides and G4PAMAM. Additionally, LDMDSs allowed us to explain the structure and energetic basis of the molecular recognition between peptides and G4-PAMAM that explains affinity and saturation at the atomic level. Our theoretical findings replicate the experimental G4-PAMAM-peptide complex stoichiometry at neutral pH, revealing that despite 8 to 9 peptides reaching a stable G4-PAMAMpeptide complex, only three peptide conformers are able to encapsulate, without impacting G4-PAMAM sphericity shape properties, which is in contrast to the saturation of G4-PAMAM by 20e30 MTX ligands previously published. Remarkably, the G4PAMAM-peptide complex is unstable at acidic pH, indicating a fast release of both peptides under acidic environment; however, this statement requires experimental validation since the only results present in the literature correspond to neutral conditions. LDMDSs coupled to the MMGBSA method using different IGB models allowed us to select the IGB1 model as the best to estimate the DGmmgbsa values between the target peptides and G4-PAMAM. The IGB1 model allowed us to identify that polar and nonpolar interactions guided the molecular recognition between ABC and PGV04 and G4-PAMAM at neutral pH, with nonpolar interactions being the major contributors to complex stabilization. Together, information about the binding properties of the two negatively
Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments The work was supported by grants from CONACYT (A1-S-21278) and SIP/IPN (20190133).
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Topical Perspectives
Complexation of peptide epitopes with G4-PAMAM dendrimer through ligand diffusion molecular dynamic simulations Correa-Basurto Martiniano Bello*, Rolando Alberto Rodríguez-Fonseca, Jose rmacos y Bioinforma tica, Escuela Superior de Medicina, Instituto Polit ~ o de fa Laboratorio de Modelado Molecular, Disen ecnico Nacional, Plan de San Luis y n, Ciudad de M Díaz Miro exico, 11340, Mexico
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 September 2019 Received in revised form 12 December 2019 Accepted 16 December 2019 Available online 20 December 2019
Peptide epitopes from HIV-1 gp120 have been used to block the gp120-CD4 complex, whereas their poor absorbable or immunogenic properties prevent them from coupling to generation four polyamidoamine (PAMAM-G4) dendrimers. PAMAM-G4 are synthetic nanoparticles that are relatively nontoxic and nonimmunogenic have been employed as nanocarriers. In a previous study, two peptide epitopes (ABC and PGV04) from gp120 located at the protein-protein interface of the gp120-CD4 complex were identified through protein-protein dissociation. Then, their complexation with G4-PAMAM was evaluated through experimental and theoretical approaches, revealing a stoichiometry of 1:8/9 for G4-PAMAM and ABC or PGV04, respectively, providing important information that can be used to gain insight into the structural and energetic basis of the molecular binding of these G4-PAMAM-peptide systems. In this contribution, we performed ligand diffusion molecular dynamic simulations (LDMDSs) using 1.5 ms combined with the molecular mechanics generalized Born surface area (MMGBSA) approach, a strategy that successfully reproduced experimentally encapsulation on PAMAM-G4-ligand complexes, to explore the mechanism through which ABC and PGV04 are encapsulated by PAMAM-G4 under neutral and acid conditions. Our results reproduce the reported PAMAM-G4-peptide complex stoichiometry, revealing a slower peptide delivery at neutral conditions and a spontaneous release under acidic conditions. LDMDSs show that several peptides can reach stable G4-PAMAM complexes at neutral pH, and only a few are able to encapsulate on dendrimers without impacting dendrimer sphericity. Energetic analysis exploring different generalized Born models revealed that the ABC peptide has better binding properties than PGV04. © 2019 Elsevier Inc. All rights reserved.
Keywords: PAMAM-G4 Dendrimer Peptide Ligand diffusion molecular dynamics simulations Binding free energy
1. Introduction The protein gp120 is the first HIV-1 glycoprotein that establishes contact with host target cells on the CD4 receptor [1]. Blocking gp120-CD4 complex formation may prevent HIV-1 infection in host CD4þ target cells [2]. Thus, three-dimensional (3D) models of the quaternary structure of HIV-1 envelope proteins (gp120-gp41) forming complexes with broadly neutralizing antibodies (bnAbs) from cryo-electron microscopy (PDB: 3J5M) are valuable resources for peptide epitopes. This gp120-gp41 complex displays a wellordered quaternary structure that permits a good antigenic profile and as native gp120-gp41 trimers, discriminates between recognition by bnAbs and non-bnAbs [3]. Thus, this structural
* Corresponding author. E-mail address: [email protected] (M. Bello). https://doi.org/10.1016/j.jmgm.2019.107514 1093-3263/© 2019 Elsevier Inc. All rights reserved.
information could be exploited to develop HIV-synthetic peptide epitope vaccines that are safer, cheaper and immune-targeted to CD4 binding sites, a vulnerability domain of HIV-1 [4]. However, challenges faced by peptide epitopes, such as instability in the presence of endogenous enzymes, poor immunogenicity and poor antigen-presenting cell (APC) uptake, limit the development of HIV-synthetic peptide epitope vaccines. These challenges could be resolved by using dendrimer-based delivery platforms. Dendrimers are branched nanoparticles used as drug-delivery approaches with high water solubility [5] and biocompatibility [6]. Dendrimers can encapsulate ligand molecules by nonbonding interactions inside their empty cavities present around the core [7]. In addition, these dendrimer platforms have previously been demonstrated to enhance the peptide-specific immune response by acting as adjuvants [8,9], enhancing antigen uptake by APCs [10] and improving the vaginal-immune response of synthetic-peptide vaccines [8,9]. However, prior to evaluating the capability of dendrimers to
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improve ligand properties of the peptide, the dendrimer-peptide complexes need to be characterized by physicochemical procedures [8]. Theoretical procedures are valuable tools for screening peptide ligands that are able to form dendrimer complexes. However, in silico characterization methods for dendrimer-ligand complexes involve small molecule ligands, and traditional methods of molecular docking have limitations mainly for aqueous environment simulations [11], which is because these methods do not consider solvation energies and algorithm limitations in peptide molecules with higher rotatable bonds or higher conformational-space [11]. Due to this limitation, docking and MD simulations have been combined to get insight into the energetic basis of the molecular recognition and encapsulation of small ligands, peptides and nucleic acids by native or modified dendrimers [13e18]. In this context, Marquez-Miranda et al. combined docking and MD simulations to evaluate the ability of G4-PAMAM-OH dendrimers to bind angiotensin peptides of seven amino acids, revealing that this dendrimer is able to bind three angiotensin peptides through docking calculations, whereas MD simulations showed that the dendrimer-peptide complex was stabilized through electrostatic and vdW interactions [12]. However, a drawback of this latter study may be the restriction of rotatable bonds for peptides higher than 7 amino acids present in the AutoDock 4.2 suite. Ligand diffusion inside proteins have been explored through MD simulations in combination with several methodological approaches that apply forces to the ligand to accelerate the binding or unbinding process [19e21]. Recently, a theoretical method named ligand diffusion molecular dynamic simulations (LDMDSs) combined with the molecular mechanics generalized surface area (MMGBSA) approach was employed to explore the mechanism through which methotrexate (MTX) is complexed to G4-PAMAM dendrimers at neutral, basic, and acidic conditions. The theoretical approach was able to reproduce experimental stoichiometry between MTX and PAMAM-G4 under neutral conditions, whereas the binding free energy value suggested a much slower release of MTX in neutral conditions compared to acidic conditions, and poor affinity was observed under basic conditions [22]. Under the LDMD approach, one of several ligands is positioned at different distances from the receptor, permitting molecular recognition through conventional MD simulations without employing any external bias that forces binding. This approach has been used to explore the complex formation between proteins and small organic molecules [23e25], whereas LDMDs in combination with the MMGBSA approach have been used to observe ligand diffusion on proteins [26] and on PAMAM-G4 dendrimers [22]. However, as far as we are concerned, this method has not been employed to assess the loading and encapsulation properties of peptides on the PAMAM-G4 dendrimer. In a previous study, experimental and theoretical approaches, including docking studies followed by MD simulations, were employed to elucidate the binding stoichiometry of two peptide epitopes (ABC and PGV04) identified from gp120 after structural dissection of the gp120-CD4 complex [27], revealing a stoichiometry of 1:8 and 1:9 for G4-PAMAM and the ABC or PGV04 peptides, respectively, providing important information about the structural complexation but lacking important structural and energetic information about the affinity and encapsulation properties of both peptides by G4-PAMAM dendrimers under neutral and acidic conditions, which represent the physiologic conditions of vaccine transportation and delivery, respectively. Therefore, this represents a good system to evaluate the ability of LDMDSs combined with the MMGBSA approach to reproduce experimental stoichiometry and provide structural and energetic information that is difficult to obtain through experimental approaches and docking studies. In
this study, we employed LDMDs together with the MMGBSA approach in the microsecond (ms) time scale using saturating concentrations of the ABC and PGV04 peptides to explore the mechanism through which these peptides are complexed with PAMAMG4 at neutral and acidic conditions to make information concerning the energetic and encapsulation properties available. 2. Material and methods 2.1. Ligand diffusion MD simulations The three-dimensional structures of the PGV04 (SFANSSGGDLEV) and ABC (GSTNSTTETFRPGGGD) peptides were taken from a previous study [8]. Each peptide conformation was placed around the charge-neutral and charge-acid G4-PAMAM at a peptide ligand to G4-PAMAM ratio of 10:1 at different distances that oscillated between 7.0 and 15 Å from the G4-PAMAM using AmberTools 16 [28]. Force field parameters and tridimensional data for chargeneutral PAMAM-G4 were taken from the website http://www. physics.iisc.ernet.in/~maiti/dbt/home.html, and the force field parameters and tridimensional data for charge-acid PAMAM-G4 were taken from a previous work [13]. LDMDSs were performed with the Amber 16 package together with the ff14SB force field [29] and the generalized Amber force field (GAFF) [30]. The topologies of the G4PAMAM-peptide complex systems were built by the LEaP module and minimized and equilibrated through the Sander module and pmemd.cuda Amber 16 [28] through the use of Graphical Units Processors [31]. Aspartic and glutamic acid amino acids of the PGV04 and ABC peptides were modeled as protonated and unprotonated for simulations under acid and neutral pH, respectively. Chlorine atoms were placed at different locations by LEaP to mimic physiological conditions and neutrality. A rectangular-shaped box of water was constructed using the TIP3P water model [32]; the water model was extended 20.0 Å between the G4-PAMAM and the limit of the box. Afterwards, the solvated and neutralized G4PAMAM was minimized and equilibrated by the following steps: minimization through 5000 steps of steepest descent minimization; 1000 picoseconds (ps) of heating; 1000 ps of density equilibration with weak restraints on the complex; and 10 ns of constant pressure equilibration at 310 K using the SHAKE algorithm [33] on hydrogen atoms to allow a time step of 2 femtoseconds (fs) and Langevin dynamics for temperature control. The equilibration run was followed by 250-ns-long MD simulations, without position restraints under periodic boundary conditions (PBC) and under an NPT ensemble at 310 K. The particle mesh Ewald method (PME) [34] was used to treat long-range electrostatic interactions under periodic conditions with a direct space cutoff of 10 Å, and a similar cutoff was employed for Van der Waals interactions. The time step of the MD simulations was set to 2.0 fs, and the SHAKE algorithm [33] was used to constrain bond lengths at their equilibrium values. Temperature and pressure were maintained using the weakcoupling algorithm [35] with coupling constants tT and tP of 1.0 and 0.2 ps, respectively (310 K, 1 atm). For each system, three independent 250-ns-long MD simulations with equal parameters were carried out. Coordinates were retrieved every 20 ps for analysis. AmberTools 16 was used to analyze the time-dependence of the solvent accessible surface area (SASA) and radius of gyration (RG) to obtain different dendrimer conformers. 2.2. Calculation of binding free energies Binding free energies of G4-PAMAM-peptide complexes were calculated using the MM/GBSA approach [36e38] provided in the Amber 16 suite. A total of 1000 snapshots were selected at each 100 ps time interval from the equilibrated simulation time for G4-
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PAMAM-peptide complexes (150-ns-long simulations). The solvation free energy (DGsol) was determined with three different generalized Born (GB) continuum solvation models implemented in Amber16: the pairwise GB model of Hawkins and colleagues (GB1) [39e41], and two modified GB models developed by Onufriev and collaborators (GB2 and GB5) [42]. The relative binding free energy (DGmmgbsa) of each G4-PAMAM-peptide complex represented the average value of three independent simulations and was determined as described previously [13].
3. Results and discussion 3.1. Equilibrium of G4-PAMAM-peptide complexes Analysis of the 250-ns-long LDMDSs between peptides and charge-acid and charge-neutral G4-PAMAM (Fig. 1) shows that the charge-acid G4-PAMAM-ABC-peptide and charge-neutral G4PAMAM-PGV04-peptide equilibrated between 10 and 25 ns with average Rg and SASA values of 24.6 ± 0.40 and 21709 ± 306.70 Å, respectively, for the G4-PAMAM-ABC-peptide system and 24.8 ± 0.37 and 21658 ± 338 Å, respectively, for the G4-PAMAMPGV04-peptide. G4-PAMAM-ABC-peptide and G4-PAMAM-PGV04peptide equilibrated between 25 and 50 ns with average Rg and SASA values of 21.14 ± 0.60 and 14035.95 ± 699.50 Å, respectively, for the G4-PAMAM-ABC-peptide system and 20.73 ± 0.44 and 12843.46 ± 605.27 Å, respectively, for the G4-PAMAM-PGV04peptide. The Rg and SASA values were in line with those previously reported [13,22,43e49]. Comparison of the Rg and SASA values of G4-PAMAM at acidic and neutral pH with those of free G4PAMAM indicates that the coupling of peptides does not significantly impact the sphericity shape of G4-PAMAM, which is in contrast to previous results in which the saturation of the G4PAMAM dendrimer with methotrexate affected the shape of PAMAM-G4 with respect to its free state at neutral conditions [22].
3
3.2. Dynamics of the complex formation The G4-PAMAM dendrimer comprises surfaces similar to pockets, where the walls and bottom of the pockets are apolar and the spikes form the edges of the pocket and are polar. At neutral pH, the primary amines are positively charged, which results in an electrostatic repulsion at the dendrimer surface, allowing the formation of a number of cavities [13]. The number of cavities is increased in acidic conditions as a result of the electrostatic repulsion between the positively charged primary and tertiary amines at the dendrimer surface and internal branches, allowing the formation of more cavities that exhibit better encapsulation properties for small organic molecules [22]. LDMDSs at 250 ns showed that from the ten ABC peptide (Fig. 2A) and PGV04 peptide (Fig. 2E) conformers around chargeneutral G4-PAMAM dendrimers, nine ABC peptides (Fig. 2B) and ten PGV04 peptides (Fig. 2F) conformations were able to form complexes with G4-PAMAM dendrimers at 50 ns. Ten ABC (Fig. 2C) and PGV04 (Fig. 2G) peptides reached stabilized G4-PAMAM-peptide complexes from 150 ns until the end of the MD simulation. On the other hand, 250 ns-long LDMDSs for the ABC and PGV04 peptides with the charge-acid G4-PAMAM showed that both peptides were unable to reach a stable conformation with G4-PAMAM (Fig. 3). Although structural analysis of the peptides coupled to neutral G4-PAMAM revealed differences in the degree of encapsulation of peptides by G4-PAMAM dendrimers (Fig. 2C and 2G) and the lack of affinity of both peptides with acid G4-PAMAM, more accurate structural analysis needs to be performed to evaluate the degree of encapsulation of peptides at neutral pH. Therefore, quantitative persistence of the G4-PAMAM-peptide complexes was evaluated by quantifying the distance between the center of mass (COM) of the ten ABC and PGV04 peptide conformations and the COM of tertiary amines from G4-PAMAM. As shown in Table 1, only conformer1, conformer2 and conformer5 of the ABC-peptides showed COM distances lower than the Rg value (21.08 ± 0.65 Å) of the free charge-neutral G4-PAMAM
Fig. 1. Radius of gyration and solvent-accessible surface area analysis of the charge-acid and charge-neutral G4-PAMAM-peptide complexes through 250 ns LDMDS together with the MMGBSA approach.
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Fig. 2. Molecular diffusion of ABC and PGV04 peptides on charge-neutral G4-PAMAM. Initial coordinates for the ABC (A) or PGV04 peptide (E) with charge-neutral G4-PAMAM dendrimers. Coordinate taken during the 50 ns-long MD simulation for the ABC (B) and PGV04 peptides (F) with charge-neutral G4-PAMAM dendrimers. Snapshot taken during the 250 ns-long MD simulation for the ABC (C) or PGV04 peptide (G) with charge-neutral G4-PAMAM dendrimers. Coordinate showing the three ABC (D) and PGV04 (H) peptides that encapsulate the G4-PAMAM dendrimer during the 250-ns-long MD simulation. The ABC and PGV04 peptides are shown in yellow spheres and G4-PAMAM dendrimers are shown in cyan spheres (nitrogen and oxygen atoms of peptides and dendrimers are depicted in blue and red color, respectively). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article).
Fig. 3. Molecular diffusion of the ABC and PGV04 peptides on charge-acid G4-PAMAM. Initial coordinates for the ABC (A) or PGV04 peptide (D) with charge-acid G4-PAMAM dendrimers. Coordinate taken during the 50 ns-long MD simulation for the ABC (B) and PGV04 peptides (E) with charge-acid G4-PAMAM dendrimers. Coordinate taken during the 250 ns-long MD simulation for the ABC (C) or PGV04 peptide (F) with charge-acid G4-PAMAM dendrimers.
dendrimer (Figs. 4 and 2D), suggesting that only these ABC-peptide conformers exhibit encapsulation and strong coupling, whereas the remaining ABC-peptide conformers showed superficial and weak coupling. Similarly, for PGV04, only three peptides were encapsulated on the G4-PAMAM dendrimer; conformer1, conformer3 and
conformer7 of the PGV04-peptides (Figs. 4 and 2H) displayed COM distances minor to the Rg value of the free charge-neutral G4PAMAM, indicating encapsulation and strong binding for these three peptide conformers, whereas the remaining PGV04-peptide conformers suggested superficial and low affinity to G4-PAMAM.
M. Bello et al. / Journal of Molecular Graphics and Modelling 96 (2020) 107514 Table 1 Distances between the center of mass (COM) of ABC or PGV04 peptide conformers and the COM of tertiary amines of G4-PAMAM. System ABC peptide Conformer 1 Conformer 2 Conformer 3 Conformer 4 Conformer 5 Conformer 6 Conformer 7 Conformer 8 Conformer 9 Conformer 10
COM 15.98 16.94 28.77 34.23 32.63 30.40 11.57 24.44 30.80 24.44
± ± ± ± ± ± ± ± ± ±
1.77 1.13 3.53 1.63 3.81 1.98 1.05 1.62 3.03 1.59
System PGV04 peptide Conformer 1 Conformer 2 Conformer 3 Conformer 4 Conformer 5 Conformer 6 Conformer 7 Conformer 8 Conformer 9 Conformer 10
COM 20.28 ± 3.04 23.84 ± 3.58 12.305 ± 1.10 22.19 ± 1.89 12.17 ± 0.90 28.53 ± 3.61 21.18 ± 2.21 12.86 ± 1.22 25.60 ± 1.07 29.27 ± 2.49
5
mer2 > conformer3 > conformer9 > conformer5 > con former4 > conformer6. Comparison of the ranking affinity employing the three GB solvation models revealed that despite the differences observed for conformer7 and conformer10 between the IGB2/IGB1 and IGB5 models, a similar tendency was observed for the coupling of the ABC peptides on the G4-PAMAM dendrimer. The ten PGV04 peptide conformers showed the following affinity ranking for the IGB1 model: conformer5 > confor mer4 > conformer7 > conformer8 > con former10 > conformer1 > conformer2 > conformer6 > con former9 > conformer3. For the IGB2 model, we identified the following affinity order: conformer5 > conformer4 > con former7 > conformer10 > conformer8 > conformer1 > conformer
Fig. 4. Center of mass (COM) of the ABC or PGV04 peptide with G4-PAMAM. Distances between the COM of ABC or PGV04 peptide conformers and the COM of tertiary amines of charge-neutral G4-PAMAM during the 250-ns-long LDMDSs.
Overall, this analysis reveals that despite the structural differences of both peptides, only three peptide conformers are able to be encapsulated by G4-PAMAM at neutral pH conditions. 3.3. Binding free energy Although structural analysis of the peptide encapsulation by G4PAMAM provides important information about the ability of small organic molecules or peptides to be transported by G4-PAMAM, it is also important to quantify the affinity of the peptides coupled to G4-PAMAM. Tables IeIII (supplementary material) show that nonpolar interactions mostly guided the molecular recognition between ABC or PGV04 and G4-PAMAM when the GB2 and GB5 models were employed, whereas the GB1 model showed that the relative binding free energy employing the MMGBSA approach (DGmmgbsa) was the consequence of the contribution of polar and nonpolar contributions which contributed mostly to G4-PAMAMpeptide complex stabilization. Comparison of the DGmmgbsa values between the ABC or PGV04 conformers and G4-PAMAM (Table 2) showed that the peptides were coupled to the G4-PAMAM surface with different affinities. The DGmmgbsa values between the ten ABC conformers with G4PAMAM exhibited the following affinity ranking when the IGB1 and IGB2 models were used: conformer1 > confor mer10 > conformer7 > conformer8 > conformer2 > conformer3 > conformer9 > conformer5 > conformer4 > conformer6. The IGB5 model showed the following rankings: conformer1 > conformer7 > conformer10 > conformer8 > confor
Table 2 Binding free energy components (kcal/mol) of G4-PAMAM-peptide complexes using the MMGBSA approach and three different continuum solvation models: GB1, GB2 and GB5. System GB1 G4-PAMAM-peptide complex ABC Conformer 1 90.38 ABC Conformer 2 45.73 ABC Conformer 3 35.89 ABC Conformer 4 30.03 ABC Conformer 5 31.47 ABC Conformer 6 11.31 ABC Conformer 7 61.76 ABC Conformer 8 50.64 ABC Conformer 9 34.53 ABC Conformer 10 65.53 PGV04 Conformer 1 47.96 PGV04 Conformer 2 40.88 PGV04 Conformer 3 37.67 PGV04 Conformer 4 64.06 PGV04 Conformer 5 98.51 PGV04 Conformer 6 40.24 PGV04 Conformer 7 58.52 PGV04 Conformer 8 58.06 PGV04 Conformer 9 38.22 PGV04 Conformer 10 54.64
(0.42) (0.43) (0.42) (0.27) (0.30) (0.24) (0.35) (0.43) (0.33) (0.41) (0.60) (0.38) (0.28) (0.36) (0.49) (0.48) (0.37) (0.31) (0.37) (0.52)
GB2
GB5
70.24 (0.35) 28.60 (0.34) 24.72 (0.34) 17.39 (0.21) 19.31 (0.22) 5.32 (0.16) 47.50 (0.31) 32.35 (0.30) 22.65 (0.24) 47.99 (0.33) 33.42 (0.45) 26.20 (0.28) 22.09 (0.23) 46.76 (0.23) 78.32 (0.43) 28.56 (0.48) 42.57 (0.30) 38.60 (0.28) 25.23 (0.29) 41.39 (0.42)
73.02 (0.38) 30.01 (0.37) 24.65 (0.37) 15.61 (0.23) 19.23 (0.24) 4.58 (0.17) 51.35 (0.35) 32.03 (0.31) 22.50 (0.26) 48.73 (0.41) 33.58 (0.47) 25.31 (0.30) 21.22 (0.28) 47.26 (0.31) 88.83 (0.49) 28.77 (0.50) 43.91 (0.33) 42.06 (0.31) 25.54 (0.32) 42.65 (0.45)
6 > conformer2 > conformer9 > conformer3. For IGB5, the following tendency was observed: conformer5 > confor mer4 > conformer7 > conformer10 > conformer 8 > conformer1 > conformer6 > conformer9 > con
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M. Bello et al. / Journal of Molecular Graphics and Modelling 96 (2020) 107514
Fig. 5. Representative G4-PAMAM-peptide conformations at neutral pH. The three G4-PAMAM-ABC-peptide conformations: conformer1 (A), conformer2 (B) and conformer5 (C). The three G4-PAMAM-PGV04-peptide conformations: conformer 1(D), conformer3 (E) and conformer5 (F) that encapsulate in the stabilized complex.
former2 > conformer3. Comparative analysis of the ranking affinity employing the three GB solvation models revealed that the IG2 and IGB5 models exhibited a more similar ranking affinity among them than with IGB2; however, the three GB solvation models were able to identify the first four peptide conformers with the highest DGmmgbsa values (conformer5, conformer4, conformer7 and conformer8/10) and were able to identify the peptide conformer coupled with the less favorable DGmmgbsa value (conformer3). Estimation of the performance of the three IGB models revealed that although the IGB2 and IGB5 models were better at reproducing similar affinity tendencies for the coupling of peptides with PAMAM-G4, the IGB1 model was more suitable since it allows the estimation of the polar and nonpolar contributors to the DGmmgbsa
value, whereas the IGB2 and IGB5 models underestimate the polar contribution. Interestingly, this result indicated that the IGB1 model was more suitable for estimating binding affinities in the PAMAM-G4-peptide/protein system, which is in contrast to previous studies for complexes between PAMAM-G4 and small organic molecules [13] and protein-ligand systems [50], where the GB2 model showed better accuracy in describing the polar and nonpolar components than GB1 and GB5. Based on the DGmmgbsa values obtained with the IGB1 model and the number of peptides coupled to G4-PAMAM, PGV04 exhibited a more favorable binding affinity for G4-PAMAM than ABC (Table 2). Comparison of the DGmmgbsa values with respect to the distances between the COM of ABC or PGV04 peptide conformers and the
M. Bello et al. / Journal of Molecular Graphics and Modelling 96 (2020) 107514
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COM of tertiary amines of G4-PAMAM (Table 1) showed that there was a relationship between the highest DGmmgbsa values observed between ABC conformation (conformer1, conformer2 and conformer7) with G4-PAMAM and low COM values. Similarly, a relationship between low COM values and highest DGmmgbsa values was observed between two PGV04 peptide conformations (conformer5 and conformer8) with G4-PAMAM. Interestingly, there was a relationship between the highest DGmmgbsa values and low COM values for some peptides but not others, as for the case of conformer3 of PGV04 (Tables 1 and 2), indicating that for the complexation of some peptides with G4-PAMAM, the peptide does not necessarily need to be encapsulated to show energetically favorable DGmmgbsa values, which is in contrast with the complexation between single small organic molecules and PAMAM-G4, where a correlation was observed between the degree of encapsulation and the binding affinity [13], but in line with LDMDS studies where G4-PAMAM was saturated with 20e30 methotrexate ligands at three different pH values [22].
charged peptides with G4-PAMAM indicates that the ABC peptide is a better candidate for use as an epitope peptide since it exhibits a lower affinity for G4-PAMAM than PGV04, allowing for easier release from the dendrimer. In addition, the in silico methodology employed in this study could be employed to reduce the cost of screening synthetic peptides capable of attaching, with stability, to G4-PAMAM to carry out experimental assays.
3.4. Structural analysis of complex formation
Appendix A. Supplementary data
Analysis of the interactions present in the three G4-PAMAMABC/PGV04-peptide and G4-PAMAM-PGV04-peptide complexes showed that both peptides were stabilized by polar interactions between polar atoms of polar and charged residues of ABC peptides with the polar primary and tertiary amines and amide groups of the PAMAM-G4 dendrimer (Fig. 5AeC). For PGV04, similar polar interactions occurred between polar, charged and hydrophobic residues with PAMAM-G4 cavities (Fig. 5DeF). Based on the participation of Asp7 and Glu9 of the ABC peptide and Asp9 and Glu11 of the PGV04 peptide in hydrogen bonding, it can be suggested that the protonated state modeled for these residues in LDMDs under acidic conditions (see methods) prevented peptide coupling on the charge-acid G4-PAMAM. However, the nonpolar interactions were the most important to stabilize the G4-PAMAMpeptide complex system, occurring between the nonpolar atoms of the peptides and the nonpolar atoms of the PAMAM-G4 dendrimer.
Supplementary data to this article can be found online at https://doi.org/10.1016/j.jmgm.2019.107514.
4. Conclusions In this study, we evaluated the ability of LDMDSs combined with the MMGBSA approach to reproduce the experimentally observed stoichiometry between the ABC and PGV04 peptides and G4PAMAM. Additionally, LDMDSs allowed us to explain the structure and energetic basis of the molecular recognition between peptides and G4-PAMAM that explains affinity and saturation at the atomic level. Our theoretical findings replicate the experimental G4-PAMAM-peptide complex stoichiometry at neutral pH, revealing that despite 8 to 9 peptides reaching a stable G4-PAMAMpeptide complex, only three peptide conformers are able to encapsulate, without impacting G4-PAMAM sphericity shape properties, which is in contrast to the saturation of G4-PAMAM by 20e30 MTX ligands previously published. Remarkably, the G4PAMAM-peptide complex is unstable at acidic pH, indicating a fast release of both peptides under acidic environment; however, this statement requires experimental validation since the only results present in the literature correspond to neutral conditions. LDMDSs coupled to the MMGBSA method using different IGB models allowed us to select the IGB1 model as the best to estimate the DGmmgbsa values between the target peptides and G4-PAMAM. The IGB1 model allowed us to identify that polar and nonpolar interactions guided the molecular recognition between ABC and PGV04 and G4-PAMAM at neutral pH, with nonpolar interactions being the major contributors to complex stabilization. Together, information about the binding properties of the two negatively
Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments The work was supported by grants from CONACYT (A1-S-21278) and SIP/IPN (20190133).
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