Role of the Bound Phospholipids in the Structural Stability of

Article Cite This: J. Phys. Chem. B XXXX, XXX, XXX−XXX

pubs.acs.org/JPCB

Role of the Bound Phospholipids in the Structural Stability of Cholesteryl Ester Transfer Protein Prasanna D. Revanasiddappa,† Revathi Sankar,† and Sanjib Senapati* BJM School of Biosciences and Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600036, India S Supporting Information *

ABSTRACT: Cholesteryl ester transfer protein (CETP) facilitates the transfer of cholesteryl esters (CEs) from antiatherogenic high-density lipoproteins to proatherogenic low-density lipoproteins. Inhibition of CETP is therefore being pursued as a potential strategy to reduce cardiovascular risk. The crystal structure of CETP has revealed the existence of two neutral CEs and two charged phospholipids (PLs) in its hydrophobic tunnel. This is in direct contrast to the other lipid-binding proteins that contain only two bound lipids. Moreover, previous animal studies on mice showed no detectable PL-transfer activity of CETP. Thus, the role of bound PLs in CETP is completely unknown. Here, we employ molecular dynamics simulations and free-energy calculations to unravel the primary effects of bound PLs on CETP structure and dynamics and attempt to correlate the observed changes to its function. Our results suggest that the structure of CETP is elastic and can attain different conformations depending on the state of bound PLs. In solution, these PLs maintain CETP in a bent−untwisted conformation that can uphold neutral lipids in its core tunnel. Results also suggest that although both PLs complement each other in their action, the C-terminal PL (C-PL) imparts greater influence on CETP by virtue of its tighter binding. Our finding fits very well with the recent inhibitor-bound CETP crystal structure, where the inhibitor displaced the N-terminal PL for binding to CETP’s central domain without disrupting the binding of C-PL. We speculate that the observed increased flexibility of CETP in the absence of PLs could play a crucial role in its binding with lipoproteins and subsequent lipid-transfer activity.

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INTRODUCTION Cholesteryl ester transfer protein (CETP) is a 74 kDa hydrophobic glycoprotein that belongs to a family of lipidbinding/-transport proteins.1,2 Some of the key members of the family include lipopolysaccharide-binding protein (LBP),3 bacterial permeability-increasing (BPI) protein,3−5 phospholipid-transfer protein (PLTP),4,6 and so forth. These proteins primarily act as transport vehicles to move water-insoluble lipids through the blood stream. However, some of the members in the family have evolved to perform diverse functions such as inflammatory responses, toxin neutralization, metabolic regulation, and so forth.4 The recent crystal structures of CETP,7,8 LBP,9 and BPI10 demonstrated that despite their low sequence homology, the three-dimensional structures of these proteins are very similar. The uniqueness of CETP lies in the fact that it facilitates the transfer of neutral lipids in plasma, unlike the other family members that predominantly carry charged lipid molecules.11 The transfer of the neutral lipids by CETP involves the bidirectional exchange of cholesteryl esters (CEs) and triglycerides (TGs) between lipoproteins. While it transports CEs from high-density lipoprotein (HDL) to low-density lipoprotein (LDL) and very low density lipoprotein (VLDL), it carries back TGs from VLDL and LDL to HDL.2,12,13 Recent © XXXX American Chemical Society

studies have shown that the deficiency of CETP in humans and rabbits results in a reduced susceptibility to the development of atherosclerosis.14−16 This lowered risk for the onset of coronary heart diseases was attributed to the inhibition of CETP, which led to the enrichment of good cholesterol, HDL-CEs, with a simultaneous depletion of the bad cholesterol, LDL-CEs.17,18 Since then, CETP has been actively pursued as a potential target for the treatment of cardiovascular diseases (CVDs).12,19 The growing interest in CETP inhibition has also led to a major research effort to understand its lipid-transfer mechanism.7,20,21 The X-ray crystallographic structure of CETP showed the protein to be an extended “boomerang”-shaped molecule with a two-domain architecture (Figure S1).7 Each of the N- and Cterminal domains consisted of a highly twisted beta barrel and two helices. The two domains are interfaced by a central beta sheet, made up of six antiparallel beta strands. The presence of a flexible linker and a C-terminal extension, which folds into an amphipathic helix, helix-X, makes CETP unique among the members of the lipid-binding/-transport protein family. CETP contains two neutral CEs, which are accommodated in the 60 Å Received: December 8, 2017 Revised: February 15, 2018 Published: March 28, 2018 A

DOI: 10.1021/acs.jpcb.7b12095 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B

maintained by adding appropriate numbers of Na+ and Cl− counter ions. An extensive set of minimization and thermalization was performed to remediate any overlap or close proximity of the added water and ions to the CETP structure and to relax CETP from its lattice-constrained conformation in the crystal. Here, energy minimization of 2500 steps was performed using the steepest descent algorithm. Later, these systems were gradually heated up to reach a temperature of 310 K using a V-rescale thermostat with a coupling constant of 0.1 ps.29 Subsequently, the solvent density was adjusted under isobaric and isothermal conditions at 1 bar at 310 K with the help of a Parrinello−Rahman barostat with isotropic pressure coupling and a coupling constant of 0.1 ps.30 All systems were then equilibrated in NPT ensemble with a time step of 2 fs for 10 ns each. The long-range electrostatic interactions were treated using particle-mesh Ewald sum with a cutoff of 1.0 nm.31 The van der Waals interactions were terminated beyond the cutoff value of 1.0 nm, and LINCS algorithm was used to constrain all bonds involving hydrogen atoms.32 The NPT equilibration phase was required for the convergence of energy components and mass density. The resultant structure from the NPT equilibration phase was used for final production run in NPT ensemble. Free-Energy Calculations. Binding free energies (ΔGbinding) of the PLs were calculated by the molecular mechanics Poisson−Boltzmann surface area (MMPBSA) method.33 For each system, the block averaged ΔG values were computed from last five independent windows of 10 ns each (i.e., final 50 ns simulation data used). The binding energy was obtained by taking the difference between the free energies of the protein−PL complex (Gcomplex), the unbound protein (Gprotein), and the unbound PL (Gligand)

long central tunnel of the protein. It also contains two charged lipid molecules, N-terminal phospholipid (N-PL) and Cterminal phospholipid (C-PL), which cap the openings at the concave side of the boomerang-shaped structure. Interestingly, the recent inhibitor-bound crystal structure of CETP showed the absence of the N-PL, suggesting the likelihood of the inhibitor entry through the CETP N-PL site.8 As the above discussion suggests, CETP possesses binding sites for four lipid moleculestwo sites for its substrates, CEs, and two other sites for the binding of two PLs (see Figure S1). This is in direct contrast to the other lipid-binding proteins, for example, LBP and BPI, which possess only two lipid-binding pockets.7−10,22 Moreover, experimental studies performed by crossing a CETP transgene in PLTP knockout (CETPg/ PLTP0) mice showed no detectable PL-transfer activity, suggesting that CETP does not mediate a net transfer of PLs.6 Clearly, the role of bound PLs in CETP is unknown. This motivated us to understand the role of these evolutionarily conserved lipids in the structural stability and activity of CETP. We carried out all-atom molecular dynamics (MDs) simulations of CETP bound to a varying number of PLs, while retaining both the CEs in CETP’s core tunnel. MD data are analyzed in detail, and the observed changes are correlated to the known information of CETP to validate the protocol used. Subsequently, a new set of findings is proposed that are difficult to be achieved experimentally. This includes bending and twisting motions of CETP in the presence and absence of PLs, change in CETP core tunnel volume when PLs were removed, relative binding strengths of N- and C-PLs, and so forth. Details are presented in the Results and Discussion section.

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METHODS MD Simulations. MD simulations of the substrate-bound CETP were initiated from the crystallographic structure of CETP with PDB ID: 2OBD (resolution 2.2 Å).7 To understand the structural role of the plugging PLs in CETP, we compared following five systems with a varying number of PLs(a) CETP with two PLs, that is, the control, crystal structure system (2PL CETP), (b) CETP without any PLs (0PL CETP), (c) CETP with two PLs restrained (R-2PL CETP), (d) CETP with N-PL alone (N-PL CETP), and (e) CETP with C-PL alone (C-PL CETP). The missing residues ALA1, SER2, LYS3, and GLY4 in the crystal structure were added, and the mutations induced in CETP for promoting crystallization residues C1A, N88D, C131A, N240D, N341D, and I405V were back-mutated with the help of Modeller 9v14.23 All MD simulations were performed using GROMOS 53A6 force field24 of GROMACS-5.0.4 simulation package along with Berger lipid parameters for CEs and PLs.25 This force field combination has been shown to significantly overestimate the interaction between lipids and proteins. As a corrective measure, following the procedure of Tieleman et al.,26 combination rules for the lipid−protein interactions are introduced in this study, as implemented in GROMACS. The hydrogens of heavy atoms were added by pdb2gmx module of GROMACS-5.0.4, and the protonation states of each amino acid were designated through H++ server.27 The prepared systems were first vacuumminimized for 1500 steps using the steepest descent algorithm. Next, using the simple point-charge water model,28 the structures were solvated in a cubic periodic box with water extending 10 Å outside the protein in all sides. The systems were neutralized, and an ionic strength of 0.15 M was

ΔG binding = Gcomplex − (Gprotein + G ligand)

(1)

The ΔGbinding values were computed using the g_mmpbsa script available in GROMACS programme. The average free energy of each entity in the right-hand side of eq 1 can be calculated using Gx = ⟨EMM⟩ − TS + ⟨Gsolv ⟩

(2)

EMM = E bonded + Enonbonded

(3)

Gsolv = Gnonpolar + Gpolar

(4)

where x represents the protein, ligand, or protein−ligand complex. Here, EMM stands for the molecular mechanics potential energy in vacuum, TS for the entropic contribution in vacuum, and Gsolv for free energy of solvation. The EMM includes the energy of both bonded and nonbonded interactions, where Ebonded consists of bond, angle, and dihedral interactions and Enonbonded includes electrostatic and van der Waals interaction. Gsolv is defined as the energy needed to transfer the ligand from vacuum to solvent and consists of polar and nonpolar components. The Gpolar contribution was estimated by solving the linearized Poisson−Boltzmann equation, and the solvent-accessible surface area (SASA) model was used to estimate Gnonpolar that assumes a linear relationship between Gnonpolar and SASA. In general, the MMPBSA method is semiquantitative and employs multiple approximations, including the lack of conformational entropy and information about the water molecules in the binding site. Several methods based on normal mode frequencies and the covariance matrix of atomic B


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RMSF measures the residue flexibility by calculating the extent of movement of each atom around its average position. Thus, a high value of RMSF for a particular region would imply that this region is extremely flexible, while regions with restricted dynamics would exhibit low RMSF values. Figure 1a shows the RMSF comparison of the systems with and without PLs. The two systems exhibit distinct differences, particularly at the Cterminal distal loops, Ω1−Ω2; N-terminal distal loop, Ω4−Ω5;

fluctuations are tested to estimate the entropy.34,35 However, all these calculations are computationally tedious and tend to have a larger margin of standard error compared to the other energetic terms. Many of the earlier studies, therefore, have neglected the entropy calculations, particularly when comparing the binding of two similar ligands to the same protein. Following the procedures of previous studies, here we omitted the entropy calculations because the molecular structure of the two PLs binding to CETP is similar, and thus, we term our binding energy values as relative binding free energy. Principal Component Analysis. The dominant motions of a dynamic protein are generally traced by principal component analysis (PCA).36 In PCA, the protein-correlated motions are reduced to a space of independent motions. First, a covariance matrix (Cij) was generated by measuring the degrees of collinearity of atomic motions of each pair of Cα atoms. The generated covariance matrix was subsequently diagonalized to yield the matrices of eigenvectors and eigenvalues. While the eigenvectors reflect the directions, the corresponding eigenvalues define the mean-square fluctuations of the collective motions. The details of the PCA are reported in the literature.37 The dominant motions of proteins were captured using g_covar, a module of GROMACS. The porcupine plots were generated using VMD.38 Data Analyses. The following programs of the gromacs simulation package were used for analyzing the simulation data: “gmx rms” was used for the calculation of root-mean-square deviation (rmsd) (backbone), “gmx rmsf” was used for rootmean-square fluctuation (RMSF) calculation (C-α), and “gmx gyrate” was used for radius of gyration (Rg) calculation (protein). In the rmsd plot, whole trajectory was considered for the analysis. In cases of Rg, RMSF, and bending and torsion angle calculations, last 250 ns data were used when the systems were very stable (same for replica simulations). For PCA, a stable 100 ns of the simulation trajectory was considered using “gmx covar” program.

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RESULTS AND DISCUSSION To begin with, we performed three independent all-atom MD simulations of CETP(i) CETP bound to PLs, (ii) CETP without PLs, and (iii) CETP bound to restrained PLs. The first and third simulations were initiated from the CETP crystal structure (PDB ID: 2OBD).7 For the second simulation, the PLs were removed from the crystal structure, and the resultant structure was equilibrated in explicit water via 10 ns MD run. Additionally, to explore the role of the individual PLs, two more simulations were performed where either the N-PL or the C-PL was retained, removing the second PL. All the above five structures were further simulated to generate 500 ns production data for each. Replica simulations were also performed to strengthen the results. Phospholipids Are Crucial in Structural Stability and Flexibility of CETP. To ensure the structural stability of the simulated systems, first we monitored the time evolution of the rmsd of CETP with respect to its crystal structure. Figure S2 shows that the rmsd of both systems converged within the first 50 ns simulation time, implying that the systems were wellequilibrated and have stabilized. The average values of rmsd of the systems with and without PLs were 0.41 and 0.52 nm, respectively. This implies that the structure of CETP could have been less stable, if the PLs were absent. To examine the loss in the stability of CETP regionwise, we subsequently computed the RMSF of CETP residues in the absence of PLs.

Figure 1. Altered flexibility and compactness of CETP in the absence of PLs. (a) Comparison of the residue-level fluctuations of CETP in 2PL CETP (black) and 0PL CETP (red) systems. Regions with the most significant changes are labeled, which include the C- and Nterminal distal loops Ω1−Ω3, Ω4−Ω6, helix-X, and the linker region. Error bars are shown for every 10th residue including the ones that exhibit the largest RMSF at each loop, for clarity. (b) Probability distribution of Rg of CETP in 2PL CETP (black) and 0PL CETP (red) systems. Standard deviations around the average distributions are shown. (c,d) Porcupine plots showing the breathing motion in 2PL CETP and its loss in the 0PL CETP system, respectively. C


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and helix-AI are located near the PL-binding site. On the other hand, C-terminal sheet, Ω1, and Ω2 are present as far as 26 Å from the PLs, depicting both the local and allosteric roles of PLs in maintaining the structural integrity of CETP. In our previous studies, we have shown that CETP has a rhythmic motion along its long axis.40 To locate this motion in the CETP crystal structure and to see how this is modulated if PLs were absent, we have performed PCA on the 2PL and 0PL systems. The porcupine plots representing the first principal motions in Figure 1c,d clearly show that the symmetric movements of the N- and C-terminal regions with respect to the central neck region in the 2PL CETP system (breathing motion, Figure 1c) become completely random in the absence of PLs (Figure 1d). The N-terminal region undergoes a large amplitude motion, while all other regions in the 0PL system exhibit no specific directional movements. To show the convergence, we performed PCA on the replica simulations, and the results are shown in Figure S3. A similar trend of symmetric versus asymmetric movements of 2PL and 0PL CETP systems was again evident. Taken together, the results clearly suggest that PLs have a crucial role on the structural stability and flexibility of CETP. To find particularly the effects of PL flexibility on CETP, next we simulated the PL-restrained CETP system where the dynamics of bound PLs in the CETP crystal structure were fully constrained (R-2PL system). The modulated structure and dynamics of CETP in the presence of these rigid PLs are shown in Figure 2. As the inset of Figure 2a shows, the R-PL system was well-equilibrated with average rmsd falling below the rmsd profile of the control 2PL system (average rmsd 0.31 vs 0.41 nm). The RMSF plot also shows reduced fluctuations of majority of the CETP residues in the R-PL system. Concurrently, PCA showed reduced dynamics with restricted breathing motion of the protein (Figure 2b). In an earlier study, we have shown that the structural plasticity of CETP is crucial for its lipid-transfer activity.40 The reduced dynamics observed here due to restrained PLs thus could affect the CETP function as well. Analysis of the time-averaged structure also showed loss of several secondary structural elements as seen in the 0PL system, though in lesser extent (Figure 2c). Overall, our results suggest that the presence of plug-in PLs is simply not to protect the hydrophobic tunnel of CETP from the solvent as believed by earlier authors but also to help CETP in maintaining its structural integrity and plasticity. N- and C-PLs Influence CETP Differently. To understand the specific role of the N- and C-PLs on CETP, we then simulated a pair of other CETP systems where either the N- or the C-PL was removed, keeping only one PL intact in CETP. Figure 3a inset compares the rmsd of N-PL CETP (i.e., N-PLretained, C-PL-removed) and C-PL CETP (i.e., C-PL-retained, N-PL-removed) with the rmsd of the crystal structure 2PL CETP system. The rmsds are calculated by superposing each snapshot onto the crystal structure to remove the rigid body translations and rotations. The average rmsd values of the N-PL and C-PL CETP systems are found to be ∼0.66 and ∼0.44 nm, respectively. This suggests that impact of the two PLs on CETP could be dissimilar. It is also interesting to note that the rmsd of the N-PL CETP system, that is, system with missing C-PL, is substantially higher than the other systems. On the other hand, the system having C-PL alone exhibits rmsd profile as same as that of the control, crystal structure CETP system. The residue-level fluctuations in Figure 3a also show that the system with missing C-PL (i.e., N-PL CETP) becomes

and at the linker region. The replica simulations also show a similar trend (Figure S3a). These distal loops are known to be responsible for HDL and LDL sensing,20 and their observed flexible nature is in tune with the previous normal mode analysis and MD simulation results.21,39−43 For example, Koivuniemi et al.21 have noted increased flexibility of several CETP regions, such as Ω1−Ω2, linker region, and so forth, in the absence of PLs. However, their extent of increase was lesser compared to this study, presumably because of the binding of CETP on lipoprotein surfaces in their studied systems of CETP−lipoprotein complexes. Overall, our results clearly indicate that several regions of CETP become highly flexible in the absence of PLs. It is plausible that the observed varied flexibility of CETP depending on the state of bound PLs is crucial in its lipid-transfer activity. For instance, while exploring the lipid exchange mechanism of CETP bound to lipoproteins, Koivuniemi et al. noted that the flexible structure of CETP helps it to bind on curved lipoprotein surfaces. Interestingly, these authors found that the headgroups of PLs move aside and form hydrophobic patches on the lipoprotein surface, indicating that PLs might leave when CETP binds to lipoproteins. Consequently, these authors simulated a CETP−lipoprotein binary complex where the bound PLs were removed from the CETP pocket. Taken together, we believe that CETP requires PLs for its structural stability when in solution. However, while approaching to its substrates HDL or LDL/VLDL, CETP can exist in a state of 0PL that has significantly higher flexibility for its smooth binding on the lipoprotein surfaces. The loss in structural stability due to the leaving of PLs in this state is compensated by its strong interactions with the lipoproteins. In other words, our results suggest that CETP in aqueous environment must possess plugging PLs to maintain the structural stability, until it finds the substrates. Upon binding the substrates, CETP removes its PLs and forms stable CETP−lipoprotein complexes to initiate the process of neutral lipid exchange. Flexibility often modulates the structural compactness of a protein. To understand the influence of PLs on the compactness of the CETP structure, we calculated Rg. The probability distribution of Rg in Figure 1b shows that the CETP structure lost its compactness significantly in the absence of PLs. To test the statistical significance of the RMSF and Rg results, standard deviations around the average distributions are shown by taking the block averages of five 50 ns windows across the final 250 ns of simulation trajectories. The replica simulation showed a similar trend in Rg with loosened-up CETP structure when PLs were taken off (Figure S3b). In comparison, Koivuniemi et al. found a compact CETP with decreased Rg values, which can be attributed to the formation of tight CETP−lipoprotein binary complexes. Further, to explore the reason behind increased flexibility and reduced compactness of 0PL CETP, we analyzed the stability of the secondary structural units of CETP. Figure S4 shows the comparison of the time-averaged structures of 2PL and 0PL systems with respect to the CETP crystal structure. The time evolution of the secondary structures, analyzed using the DSSP module in GROMACS, is shown in Figure S5. From these figures, it is evident that the 0PL system has lost many secondary structural units, especially in the C-terminal domain. However, minimal distortion was observed in the 2PL system. Regions that exhibited the major distortions in the 0PL system include helixX, helix-A, neck region, C-terminal sheet, Ω1, Ω2, helix-BI, and helix-AI. Out of these, helix-X, helix-A, neck region, helix-BI, D


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Figure 2. Restricted dynamics and loss of secondary structures in PLrestrained CETP. (a) Comparison of the residue-level fluctuations of CETP in 2PL CETP (black) and R-2PL CETP (red) systems. The inset shows the time evolution of backbone rmsd of CETP in 2PL (black) and R-2PL (red) systems relative to the crystal structure. Error bars are shown for every 10th residue including the ones that exhibit the largest RMSF at each loop, for clarity. (b) Porcupine plot showing the reduced breathing motion in the R-2PL system. (c) Time-averaged conformation of R-2PL CETP shows loss in secondary structures relative to the control 2PL CETP (regions that have undergone transitions are highlighted in red).

somewhat less stable compared to the other CETP systems. A majority of the functionally relevant regions, for example, loops Ω1−Ω6 that are known to be important in lipoprotein binding/sensing, exhibited unexpectedly high fluctuations. On the contrary, the presence of C-PL alone (C-PL CETP) could almost reproduce the RMSF profile of 2PL CETP. Figure 3b shows the distribution of Rg of all the simulated CETP systems. From the figure, it is evident that the N-PL system loses its structural compactness significantly with a very wide distribution of its Rg. In fact, the Rg of this system resembles that of the 0PL system closely. On the contrary, the C-PL system shows a distribution that is not very dissimilar from the 2PL CETP system and well-overlaps with the R-2PL system. The replica simulation in Figure S6 showed a similar trend. In consistent with the above results, the first principal motion from the PCA reproduced the CETP breathing motions in CPL CETP, showing concurrent movements of the N- and Cterminal domains as exhibited by the control 2PL CETP system. On the other hand, the absence of C-PL (N-PL system) completely randomized the essential motions of the protein with N-terminal, showing large amplitude movements, while other regions exhibit no specific directionality (Figure 3c). This is very similar to the essential motions of CETP domains exhibited by the 0PL system. To show the

Figure 3. Changes in CETP when bound to one of the two PLs. (a) Comparison of the residue-level fluctuations in 2PL CETP (black), NPL CETP (orange), and C-PL CETP (green) systems. Regions with the most significant changes are labeled. Error bars are shown for every 10th residue including the ones that exhibit the largest RMSF at each loop, for clarity. The inset shows the backbone RMSD of the same three systems relative to the crystal structure. (b) Probability distribution of Rg of CETP in 2PL, N-PL, and C-PL CETP. The distributions for R-2PL (black dashed) and 0PL CETP (red) are also shown. Standard deviations around the average distributions are shown. (c) Porcupine plots showing the loss in breathing motion in the N-PL system but its retention in the C-PL CETP system.

convergence, we performed PCA on the replica simulations, and the results are shown in Figure S7. Taken together, it is quite imperative to conclude that the bound C-PL in the CETP C-terminal plays the primary role in maintaining the structural integrity of CETP that is important for its function, while the N-PL in N-terminal supplements the effects. PLs Help CETP Attaining Different Conformations. To further assess the impact of PLs on CETP, we checked the E


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The Journal of Physical Chemistry B deviations in CETP bending and twisting angles in the simulated systems. The bending angle was computed by ⎯→ ⎯ ⎯→ ⎯ measuring the angle between two vectors BA and BC , where B corresponds to the center of mass (COM) of the CETP central β-domain. This domain is composed of resid 10−16, resid 211−214, resid 227−238, resid 260−266, resid 442−451, and resid 454−462. From this point B, a vector was drawn to the COM of the CETP N-terminal beta-barrel domain (resid 43− ⎯→ ⎯ 154), which was designated as vector BA . Similarly, another ⎯→ ⎯ vector BC was drawn from the COM of central β-domain to the COM of the C-terminal β-barrel domain of CETP (resid 320−396). The COMs were computed using the backbone atoms of the protein and are shown in Figure 4a inset. The angle was calculated by the vector dot product.40 This can be mathematically expressed as ⎯ ⎯→ ⎯ ⎯→ ⎯ ⎯→ ⎯ ⎯→ θ bend = cos−1{(BA ·BC)/(|BC||BC|)} (5) Figure 4a shows the probability distribution of the bending angle of 2PL CETP (black) and 0PL CETP (red) systems obtained from the block analysis of five 50 ns long windows along the final 250 ns simulation trajectory. The profile shows a nearly Gaussian distribution of the CETP angle in both systems. The distribution peaks for the respective system were found at 139° and 151°. This implies that CETP would have transformed to a more linear conformation, if the PLs were absent. The replica simulations on these systems also have shown a similar trend with a minute deviation of ±3° in the distribution peaks (Figure S8a). We have also performed a similar analysis on the systems where either PL was retained. Results show that N-PL CETP attains an extended conformation as same as that of the 0PL system with the distribution peak at 149° (orange). On the other hand, the CPL bound system attains a bent conformation that resembles the bending characteristics of the 2PL CETP system, with the distribution peak at 140° (green). We will extend this discussion in the next section. We also have examined the twisting−untwisting motions in CETP by calculating its twist angles. Twisting was quantified by measuring the rotation of N- and C-terminal β-barrel domains with respect to the central β-barrel. The central β-barrel consists of six beta sheets running antiparallel to each other COM of first three sheets was considered as point A (resid 10− 16, resid 211−214, and resid 227−238) and COM of last three sheets was considered as point B (resid 260−266, resid 442− 451, and resid 454−462). The COM of the N-terminal β-barrel domain (resid 43−154) and the COM of the C-terminal βbarrel domain (resid 320−396) were designated as point C and point D, respectively (Figure 4b inset). Next, four vectors were ⎯→ ⎯ ⎯→ ⎯ defined: point A to point B as AB, point A to point C as AC , ⎯→ ⎯ ⎯→ ⎯ point B to point A as BA , and point B to point D as BD. Using ⎯→ ⎯ ⎯→ ⎯ two vectors AB and AC , the first plane X was defined, and with ⎯→ ⎯ ⎯→ ⎯ vectors BA and BD, the second plane Y was defined. The twisting angle (θtwist ) was computed by measuring the angle ⎯→ ⎯→ between the normal vectors P1 and P2 of these two planes as shown below ⎯→ ⎯→ ⎯→ ⎯→ θtwist = cos−1{(P1·P 2)/(|P1||P 2|)} (6)

Figure 4. Bending and twisting motions in CETP. (a) Probability distribution of bending angle of CETP in 2PL (black), 0PL (red), NPL (orange), and C-PL (green). Standard deviations around the average distributions are shown. The inset shows the COMs of the three distinct regions in CETP based on which angle was calculated. (b) Probability distribution of twisting of CETP in the four systems. Standard deviations around the average distributions are shown. The inset shows the COMs of the four distinct regions in CETP based on which dihedral angle was calculated. (c) Volume of hydrophobic tunnel as a function of time is shown for 2PL (black), 0PL (red), R2PL (dotted cyan), N-PL (orange), and C-PL (green).

The distributions were found to peak at 5° and 15°. Notably, the reported bending and twisting angles in the CETP crystal structure were 139° and 6°, respectively.40 This indicates that the most probable conformation of CETP from our 2PL CETP system resembles the crystal structure conformation very well, while the most probable conformation from the 0PL system simulation is a linear CETP that is about 10° more twisted than the crystal conformation. Interestingly, the results from N-PL and C-PL CETP simulations show that N-PL CETP (orange) twists with a distribution peak at 11° and that the C-PL system

Figure 4b shows the probability distributions of the angle of twisting of 2PL CETP (black) and 0PL CETP (red) systems. F


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Figure 5. Binding free energy of PLs in the CETP-binding sites. (a) Time-averaged binding energy of N-PL and C-PL in the 2PL CETP system (red). Also shown is the time-averaged binding energy of N-PL in N-PL CETP (blue) and C-PL in C-PL CETP (green). Contribution of the individual CETP residues in binding (b) N-PL and (c) C-PL is calculated.

affect the tunnel architecture, we have computed the volume of the CETP core tunnel in the presence and absence of PLs. The program trj_cavity47 as implemented in GROMACS has been used for characterization of cavities across the MD trajectory. The program exploits a rapid, grid-based approach, which enables efficient identification and analysis of protein cavities in all six positive/negative directions along the x, y, and z-axes. Figure 4c shows the time evolution of the CETP tunnel volume in the presence and absence of PLs. In the absence of PLs, a marked reduction in the CETP tunnel volume by about 1500 Å3 was observed that persisted throughout the simulation. This reduction could be a direct consequence of the twisting of the CETP structure, as shown in the previous section. We also observed a reduction in the tunnel volume in the absence of either of the PLs, as depicted in Figures 4c and S9. The central role of the hydrophobic tunnel on the rate of neutral lipid transfer has already been demonstrated. We speculate that the larger volume of the CETP hydrophobic tunnel in the presence of both PLs corresponds to an open conformation through bending and untwisting of the CETP structure, which could facilitate a faster rate of CE/TG transfer. Binding of N- and C-PLs to CETP Is Inherently Different. To underscore the reason behind the differential effects of the two PLs on CETP structure and dynamics, we

(green) twists at a much lesser distribution peak at 5°. This means that CETP with N-terminal PL alone succumbs to a linear conformation that is largely twisted, while it continues to be bent and untwisted in the presence of the C-PL. The replica simulation in Figure S8 showed a similar trend in a twisting angle of CETP. As we move to the next section, we will see that linear−twisted CETP narrows down its core tunnel, which could hamper the CE/TG transfer across CETP. Nevertheless, it is clear from the above discussion that the structure of CETP is elastic and can attain different conformations. This might have direct implications on the lipid-transfer activity of this protein. For example, the flexible structure can bend/unbend, twist−untwist depending on the curvature of the lipoprotein surfaces on which it will bind for shuttling the lipids. While for binding on the HDL surface it would bend more than the crystal conformation, it might unbend to a more linear conformation for binding on the LDL or VLDL surfaces. PLs Modulate CETP Core Tunnel. As the crystal structure of the substrate-bound CETP shows, the protein possesses a long continuous tunnel that traverses from the N- to the Cdomain.7 The substrate molecules, CEs and TGs, traverse between various lipoprotein particles through this tunnel, and the tunnel volume plays an important role in substrate transfer.20,44−46 To understand if the absence of PLs could G


Article

The Journal of Physical Chemistry B examined their relative binding strength in CETP. Figure 5a compares the binding strength of N- and C-PLs in the 2PL CETP system computed via the MMPBSA method. The average binding energy of C-PL was found to be −27.38 kcal/ mol compared to the binding energy value of −16.54 kcal/mol for the N-PL in this system. The binding strength of individual PL in N-PL and C-PL systems was estimated to be −27.12 and −14.79 kcal/mol, respectively. These binding energy values indicate not only that C-PL is bound to CETP tunnel more tightly but also that there is a cooperative effect, particularly that of the C-PL on N-PL binding to CETP. We presume that this tighter binding of the C-PL manifests in its greater influence on the CETP structure and dynamics than does the N-PL. Our results corroborate very well with the recent crystal structure of the inhibitor-bound CETP, where the inhibitor is found to displace the N-PL for binding to the CETP central domain, leaving C-PL intact in its original binding site in CETP.8 To obtain a deeper understanding of the observed differences in the binding energies, the residue-level contribution to the total interaction energy was computed for both the PLs, and the results are shown in Figure 5b,c. The figure shows that the interaction of each PL with the respective CE is not very different. However, the interaction of the CETP-lining residues with N-PL and C-PL differs drastically. A significantly larger number of CETP residues involve in interaction with the C-PL than with the N-PL. In both instances, however, majority of the interacting CETP residues were found to be hydrophobic. This can be ascribed to the fact that PLs bind to the CETP’s hydrophobic tunnel primarily through their alkane tails.7 As the figure shows, the binding of C-PL is facilitated predominantly by hydrophobic residues, ILE205 from helix-B; PHE270, LEU273, ALA274, ALA277, and PHE278 from helix-AI; LEU283, LEU285, VAL323, ILE331, and VAL344 from the sheets of C-terminal domain; VAL421, LEU425, VAL428, PHE429, LEU432, and MET433 from helix-BI; LEU206, and ILE211 from loop regions and by a few hydrophilic residues ARG424 from helix-BI and SER207, ASP208, ASP280, ARG282, and CYS325 from loop regions. The binding of NPL involves the hydrophobic interactions with residues LEU20, LEU23, and VAL30 from helix-A; MET194, PHE197, VAL198, and ALA202 from helix-B; LEU261, PHE263, PHE461, and PHE463 from the neck region; LEU467, LEU468, and PHE471 from helix-X; GLY437, VAL438, LEU440, and PHE441 from helix-BI and also a strong hydrophilic interaction with ARG201, LYS439, and SER439. Among these, the interaction of N-PL with Arg 201 through a salt bridge has already been seen in the substrate-bound crystal structure of CETP.7 Thus, the differential binding of the two PLs can be directly correlated to the intrinsic architecture of the CETP core tunnel.

although both PLs complement each other in their action, the C-PL imparts greater influence on CETP by virtue of its tighter binding. The obtained tighter binding of the C-PL fits very well with the more recent inhibitor-bound CETP crystal structure, where the inhibitor displaced the N-PL for binding to CETP’s central cavity without disrupting the binding of C-PL.8 We speculate that the observed increased flexibility of CETP in the absence of PLs can have a direct implication on its binding on the lipoprotein surfaces. Our study paves a way to conceive experiments to investigate the role of these bound lipids on the CETP structure and function more directly. For example, timeresolved fluorescence spectroscopic techniques can be employed to examine the differential flexibility of the CETP distal loops.48,49 More advanced techniques such as surface plasmon resonance techniques can be used to quantify the relative difference in the binding energy of the N- and C-PLs.50 This study and proposed future work directed toward the understanding of CETP’s lipid-transfer mechanism and its modulators could have great implications on CVD therapeutics.

CONCLUSIONS To summarize, by employing a series of unrestrained all-atom MD simulations and PCA, we explored the role of bound PLs on the structure and dynamics of CETP. Our results suggest that the presence of plug-in PLs is simply not to protect the hydrophobic tunnel of CETP from the solvent as believed earlier but also to help CETP in maintaining its structural integrity and plasticity. These lipids bind sufficiently tightly and maintain CETP in a bent and untwisted conformation in solution. This bent−untwisted CETP upholds a wide tunnel across its core that could facilitate the transfer of CEs and TGs between different lipoproteins. Results also suggest that

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.7b12095. Crystal structure of CETP; RMSD of CETP in 2PL and 0PL CETP; comparison of RMSF, Rg, and PCA of replica 2PL and 0PL CETP systems; transformation of several secondary structural elements in the 0PL system; secondary structure comparison of 2PL and 0PL; comparison of the Rg of replica 2PL, 0PL, N-PL, and C-PL CETP; comparison of the PCA of replica N-PL and C-PL CETP systems; angle distribution of CETP in the replica systems; and comparison of the hydrophobic tunnel volume in replica systems (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +91-44-2257-4122. ORCID

Sanjib Senapati: 0000-0002-6671-8299 Author Contributions †

P.D.R. and R.S. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Department of Science and Technology (DST), Govt. of India for financial support and the Computer Centre, IIT Madras for providing the computing facility.

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REFERENCES

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Role of the Bound Phospholipids in the Structural Stability of

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