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Conformational Plasticity in an HIV-1 Antibody Epitope P. R. Tulip,*,† C. R. Gregor,† R. Z. Troitzsch,† G. J. Martyna,‡ E. Cerasoli,§ G. Tranter,§ and J. Crain†,§ School of Physics, The UniVersity of Edinburgh, Mayfield Road, Edinburgh, EH9 3JZ, U.K., IBM T.J. Watson Research Center, Yorktown Heights, New York, 10598, and National Physical Laboratory, Hampton Road, Teddington, TW11 0LW, U.K. ReceiVed: January 31, 2010; ReVised Manuscript ReceiVed: April 20, 2010
The structure of a short fragment of the human HIV-1 membrane glycoprotein gp41 has been examined using a combination of parallel tempering molecular dynamics (PTMD) and far UV circular dichroism spectroscopy. The aim is to resolve conflicting reports on the solution state conformational bias in this membrane proximal domain spanning the epitope for the 2F5 monoclonal antibody. We conclude that gp41659-671 exhibits conformational plasticity in which competing folding propensities are present and can be influenced by local microenvironment. Contrary to previous reports, the 310 helix does not emerge as a dominant motif from either simulation or experiment, and this peptide is therefore not a model system for this fold type. Other fold groups such as turn motifs are identifiable at elevated temperatures in the PTMD trajectories and are potentially relevant in antibody binding. Helical populations in pure water are significantly overestimated according to the CHARMM parametrization. However, circular dichroism (CD) data show that helices are promoted in membrane mimetic solvents. As this is a membrane proximal peptide, the helical motif may well have physiological significance. 1. Introduction and Motivation Infection by the human immunodeficiency virus (HIV-1) occurs via the combined action of two envelope glycoproteins, gp120 and gp41. The gp120 protein is responsible for initial host cell recognition and interaction with target CD4 and CCR5/ CXCR4 chemokine receptor sites. Structurally, the main features of the extracellular domain of gp41 consist of a glycine-rich fusion domain at the N-terminus followed by two highly conserved helical regions (N- and C-heptad repeats) running into the membrane proximal domain. Upon insertion of the fusion peptide into the target cell, the two helical domains fold to form a 6-helix bundle with an open central channel that allows entry of the viral nucleocapsid into the host cell cytoplasm. Understanding of these early molecular-level infection events has made it possible to develop therapeutic strategies based on their inhibition as an alternative to treatment of infected cells. A key step is the elucidation of the structure and function of important molecular fragments of these proteins that can be exploited to develop vaccines. For example, in the case of gp41, deletions or mutations in the tryptophan-rich C-terminal (pretransmembrane) segment can prevent fusion.1,2 This region also spans the synthetic peptide T-20 (gp41638-673) derived from the C-terminal heptad repeat: referred to in the literature by various names including Fuzeon and EnfuViritide, it is the first FDAapproved fusion inhibitor targeting gp41. Its mechanism is still debated, but current thinking suggests it binds competitively to the N-helix and forms a steric blockade of the conformational changes required for infection.3 A second strategy in vaccine development is to elicit broadly neutralizing antibodies that target conserved epitopes of the gp120 or gp41 glycoproteins. The membrane proximal domain * Corresponding author. E-mail:
[email protected]. † The University of Edinburgh. ‡ IBM T.J. Watson Research Center. § National Physical Laboratory.
gp41659-671 (containing a segment of T20) also spans the complete epitope ELDKWA for the 2F5 monoclonal antibody4 which shows high binding affinity for this peptide.5 The affinity is reduced after binding between gp120 and CD4 suggesting that the gp41659-671 epitope is solvent exposed in the prefusogenic form but becomes less accessible or restructured after fusion.1,6 The solution structure of this peptide (in prefusogenic form) is therefore recognized to be important for the development of peptide antigens. Recently, in two separate studies, nuclear magnetic resonance (NMR) has been used to investigate the structure of the peptide gp41659-671 in aqueous solution.1,7 In the first one,1 the 1H NMR measurements, conducted at pH 7.7, revealed a series of dNN(i, i + 1) and dRN(i, i + 3) NOE connectivities running from residue E662 to the end of the peptide. The data imply that this peptide has a conformational bias toward a 310 helix in water. Random coil conformers were identified as a minor species. The authors also suggest that because this region appears to fold in the absence of tertiary contacts it is likely to be a good description of the structure of the corresponding segment in gp41. These conclusions were not supported in work by Barbato et al.,7 in which NMR measurements do not show any major population of 310-helical conformers. These authors’ analysis indicates that a mixture of conformers is present. They also tested the acetylated form of the 13-mer in a competitive binding assay using the 2F5 neutralizing antibody, and they found the affinity of the acetylated peptide to be 20-fold lower than the nonacetylated one. Because the capping of a peptide sequence stabilizes the helix, they deduce that the helix conformation is not the most relevant for binding 2F5. In addition to the potential biochemical interest in this and related peptides for fusion inhibitor design, the recent reports that gp41659-671 forms a 310-helix in water are of considerable fundamental interest. Examples of short aqueous peptides that show well-defined secondary structure in high populations are
10.1021/jp100929n 2010 American Chemical Society Published on Web 05/21/2010
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Figure 1. Initial structures employed in these simulations: (a) R-helix, (b) type-I β-turn, and (c) inverse γ-turn. In each case, the secondary structure is indicated using colored ribbon cartoons. Purple denotes an R-helix, with cyan denoting turn structures. White denotes coil-like secondary structures. Residues without the epitopal region are faded.
relatively few. One well-known exception is the 13-residue segment of ribonuclease A, which is R-helical in water at low temperature. The observation of a 310-helix is particularly surprising: This motif is relatively rare and considered less stable than the R-helix. The NMR solution structure of the peptide gp41659-671 reported in reference 1 makes it the first watersoluble nonsynthetic peptide to form a stable 310-helix in water. UVRR data8 on this system implied a wider variety of local motifs than is evident in either of the NMR measurements.1,7 Using the spectra of the backbone amide III bands as a reporter of secondary structure, these authors find evidence for significant populations of β-turn motifs as well as pPII, 310-, and π-helices. Only small populations of R-helices are inferred from the spectra. Moreover, the spectroscopic evidence implies that the relative populations of folded vs unfolded conformations show an unexpectedly weak dependence on temperature,8 which has been taken to imply that these differing conformational states are equally occupied. It should be noted that available circular dichroism data are also not in agreement over the 310 propensities: The data of Biron et al.1 imply a higher degree of 310 helical content than those of Ahmed and Asher8 and Barbato et al.7 This system has been the subject of relatively little simulation work. A molecular dynamics study by Martins do Canto et al.9 found that the gp41 peptide does not adopt a stable structure in aqueous solution, instead forming a dynamic equilibrium of interchanging globular structures. Additional members of the conformational ensemble include R-helices and turns (of unspecified type), with the rare occurrence of both 310- and π-helices. Recent work by Lapelosa et al.10 has investigated the conformational preferences of the ELDKWAS peptide using classical atomistic parallel tempering (replica exchange) MD simulations in conjunction with an implicit solvent model. These authors’ results suggest that the peptide interconverts in solution between R-helical and type-I β-turn conformations, thereby indicating a highly flexible peptide exhibiting a degree of plasticity compatible with its biological function.
Finally, the epitope region spanning 662-667 is reported to form a type-I β-turn crystalline complex with the Fab fragment of the monoclonal antibody 2F5 (World Intellectual Property Organization patent WO-00/61618). Given the contradictory nature of the data pertaining to this peptide, it is therefore of interest to perform a thorough computational and experimental study of its structural propensities. 2. Computational Methods 2.1. Configurational Sampling and Conformational Stability. To investigate the conformational landscape of the gp41 peptide and the stability of the motifs reported by Biron1 and Barbato et al.,7 we set up three simulations, each of which comprised one gp41 molecule (sequence ELLELDKWASLN, with zwitterionic terminii) and 1853 water molecules. The three starting structures employed in these simulations comprised an R-helix, a type-I β-turn, and an inverse γ-turn and may be seen in Figure 1. In each simulation, the molecules were arrayed on a cubic lattice with random molecular orientations in a cubic box of length 38 Å. The rigid, nonpolarizable, three-site TIP3P empirical force field was used to describe the water molecules, with the CHARMM22 force field15 being employed to describe the peptide. The PINY simulation package16 was employed to perform the simulations, and a cutoff distance of 10 Å was applied to nonbonded Lennard-Jones interactions, with a longrange analytical dispersion correction applied. The system was equilibrated at 300 K in the canonical ensemble for 300 ps with a time-step of 0.1 fs to anneal out unphysical contacts. Nose´-Hoover chain thermostats17 of length 2, with a relaxation period τ ) 25 fs, were employed. In each simulation, the peptide was “massively” thermostatted; i.e., each degree of freedom possessed a thermostat, while the (rigid) water molecules were each assigned an individual “molecular” thermostat. The system was then run for 500 ps with a time step of 1.5 fs in the isothermal-isobaric ensemble (using the Martyna-Tuckerman-
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Klein18 barostat) to allow for spatial relaxation. Periodic boundary conditions were employed, and Ewald summation was used to evaluate long-ranged interactions. The Ewald “alpha” parameter, which governs the width of the screening Gaussian charge distribution, was 4.16 Å-2, while the reciprocal space component was expanded up to a maximum reciprocal space vector of 14 Å-1. Atomistic simulation of even relatively short peptides such as the system of interest here is computationally challenging because they exhibit rugged energy landscapes which are not efficiently sampled by conventional molecular dynamics simulation. The basic idea of replica exchange is now well established and has been reviewed by a number of authors.19 In the method, N independent replica systems are simulated, each at a different temperature. The lowest temperature system typically corresponds to the one of physical interest. Phase space sampling may be poor at this temperature but will improve as the temperature of the replicas is increased. Sampling efficiency at the target temperature is enhanced in the simulation process by allowing pairs of replicas at different temperatures to exchange configurations, thereby improving the phase space accessibility at the target temperature. The exchange occurs with probability determined by the modified Boltzmann criterion of Coluzza and b))/(1 + exp(∆β∆U(r b))))]. Frenkel,20 P ) min[1, (exp(∆β∆U(r This is symmetric and, in common with the more commonly presented Boltzmann criterion, preserves detailed balance. Preliminary, conventional (i.e., not replica exchange simulations) simulations indicated that the type-I β-turn structure was not a stable solution motif, the turn being annealed away during the initial equilibration procedure to yield an unstructured peptide over the epitope region and a helical structure in the N-terminal region. Similar simulations demonstrated that the R- and inverse γ-turn structures were stable over the course of a 3.5 ns production run and that, furthermore, they were energetically competitive. Consequently, an array of 60 temperers was constructed, in which R-helical and inverse γ-turn structures were interleaved; i.e., temperers with an even index were occupied by a turn-like structure, with odd-labeled temperers being occupied by the helical structure. This strategy allows us to eliminate any bias that may arise owing to choice of starting configuration and permits the competition between helical and turn structures to be investigated. The temperers were spaced according to a geometric progression over the range 275 < T < 1500 K; such a large temperature range is necessary owing to the rugged free-energy landscape of this peptide. A conventional MD simulation carried out at 1500 K demonstrates that this temperature is sufficiently high to allow interconversion of the peptide between turn-like, helical, and denatured structures, indicating that the highest-temperature walker will be able to fully traverse the phase space and that our choice is therefore justified. Configuration swaps were attempted every 50 time steps, with a time step of 2 fs being employed. Data were collected over an acqusition time of 4 ns. Our replica exchange simulation therefore required a total amount of simulation time equivalent to a conventional MD simulation of length 0.24 µs. In the Supporting Information, S1, we discuss the details and efficacy of the parallel tempering array in more detail. 3. Experimental Methods ELLELDKWASLWN was purchased from Cambridge Peptides with a stated final purity of 100%. The peptide stock was prepared by dissolving the powder in water, and its solubilization was aided through addition of a few microliters of NaOH 1 M.
Tulip et al. The absorbance at 350 nm was monitored to check for complete dissolution. The concentration of the stock solution was determined spectrophotometrically using the calculated extinction coefficient at 280 nm of 14 000 M-1 cm-11 due to the presence of two tryptophan residues in the peptide sequence. The circular dichrosim (CD) solutions were prepared from the peptide stock: the solutions in buffers (20 mM sodium phosphate pH 6.6 and 20 mM ammonium acetate pH 7.7) were prepared using 0.2 M buffer stocks at the stated pH, while the samples in water were prepared by adjusting the pH, after peptide dissolution, using HCl. The concentration was then checked spectrophotometrically. The pH of each solution was measured using a microelectrode prior to the measurement. For the experiments using trifluoroethanol (TFE), the peptide stock was diluted in phosphate buffer and in buffer containing various concentrations of TFE (10%, 20%, 30%). Circular dichroism (CD) spectra were recorded on a Jasco J-810 spectropolarimeter (Japan Spectroscopic Co., Tokyo) fitted with a Peltier unit for temperature control. For all of the experiments, the concentration of peptide in solution was 0.04 mM, and the spectra are the average of three accumulations in step mode (data pitch of 1 nm, response time of 8 s, bandwidth of 1 nm) acquired in rectangular cuvettes of 0.1 or 0.2 cm path length as stated. To study the temperature dependence, CD spectra were acquired in the 5-95 °C range at 10 °C intervals. The temperature of the sample was raised at a rate of 1 °C min-1, and the solution was left for 3 min at the target temperature to equilibrate before spectra acquisition. For every sample, an appropriate solvent spectrum was recorded under identical conditions and subtracted from the corresponding sample spectrum. The data collected are expressed in molar ellipticity (deg cm2 dmol-1) for a direct comparison with the CD spectra reported to date.1,7 4. Computational Results 4.1. Conformational Analysis. These results are generated using the CHARMM force field and thus, given sufficient sampling, are limited by the ability of CHARMM to describe the gp41-water system. Our detailed conformational analysis of the parallel tempering simulations is based upon two initial conditions: the helical structure and the inverse γ-turn. In the Supporting Information, S1, we discuss the principal features of the Ramachandran plot for the turn structure. In Figure 2(a), we present the Ramachdran plot determined for the 300 K replica. It is immediately apparent that the dihedral angles display a strong preference for right-handed R-helical conformers. This is striking, given the initial turn-like structure at this temperature. Traces of this initial structure may be seen in the faint, diffuse features in the β and left-handed R-helical regions. Visualization of the simulation trajectory reveals that the 300 K replica is occupied by a helical structure within 0.5 ns; this structure is introduced through replica exchange moves. Once this structure has been introduced, it is found that it is stable over the course of the simulation, suggesting that helical structures are preferred to turns at this temperature. Bond probability plots21 offer a means to characterize the nature of the helical structure in more depth. In Figure 2(b), we present the bond probability distribution functions characterizing 310-helix formation. While overall we see a broadly uniform bond distribution as a function of residue, suggesting a strongly ordered, relatively inflexible peptide along its entire length, some distribution of bond lengths of length greater than 4 Å is observed; this is indicative of the initial turn structure. Residues 3-12 show peaks in their distribution functions in
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Figure 2. Results for 300 K. (a) Ramachandran plot at 300 K. This displays a histogram of dihedral angles for the 11 central residues. Note that these data come from our parallel tempering simulation. (b) i, i + 3 bond probability distribution functions for the gp41 peptide at 300 K. In each case, we examine the hydrogen bond between the backbone oxygen in residue i and the backbone hydrogen in residue i + 3. Note that the distribution function is normalized such that it integrates up to unity. In (c) we display i, i + 4 bond probability distribution functions, while in (d) we display i, i + 5 bond probability distribution functions.
the range 2.46-2.95 Å. Taking a separation of 2.5 Å as the hydrogen bonding threshold, all residues appear to possess some 310 content, although only residues 3-6 and 8-11 possess peaks in their bond distributions at distances less than the hydrogen bond threshold, suggesting that these residues possess the strongest predilection for participating in 310 helices. Both terminals of the peptide exhibit a greater degree of flexibility; in particular, the large spread of bond lengths for the residues 2-5 indicates that the N-terminus of the peptide exists in more extended conformations. Visual inspection of the MD trajectories reveals occasional hydrogen bonding between side chain atoms. These results indicate that the epitope region exists in an ordered, helical state. In Figure 2(c), bond probability distributions are presented for the i, i + 4 hydrogen bonds, i.e., for R-helices. It is apparent that the N-terminus displays signs of extended structure. This, combined with the distribution of bond lengths present for r < 4 Å, suggests a level of flexibility consistent with what was
inferred from the i, i + 3 bond distribution functions. Beyond the N-terminus, the distribution functions reveal a uniform, tight R-helix, which spans the epitope region (residues 4-9). The distribution functions suggest that the R-helix is the dominant structural motif, although elements of the 310-helix structure do appear to be present. Some evidence of more extended conformations (corresponding to the initial turn) is found, as may be seen in the small peaks in the distribution functions between 6 and 9 Å. In Figure 2(d), bond probability distributions for i, i + 5 bonds are illustrated. These demonstrate that there is no π-helical content in the simulation. We also examine the temperature dependence of the secondary structure elements. In Figure 3(a), the Ramachandran plot as determined at 400 K is presented. This displays a marked similarity to the 300 K plot, with less weight appearing in the left-handed R and β regions of the plot, suggesting that there is less turn-like content at this temperature. In Figures 3(b) and 3(c), analogous bond distribution plots are
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Figure 3. Results for 400 K. (a) Ramachandran plot. (b) i, i + 3 bond probability distribution functions. (c) i, i + 4 bond probability distribution functions.
presented indicating the conformational propensities of the peptide at 400 K. These indicate the stability of the helical structure at this temperature and suggest differences in the flexibility of the N-terminus. At 844 K (40th replica), an increasingly disordered structural picture is found, in which the peptide exhibits increased flexibility. In Figure 4, the Ramachandran plot for this replica is presented, and it may be seen that the peptide is able to sample more dihedral space than is possible at lower temperatures. Analysis using STRIDE22 indicates the existence of an ensemble of conformers at the residue level for this replica that includes unordered coil, 310-, R-helical, and turn structures. It is instructive to compare our results to recent MD studies by Martins do Canto et al.9 and Lapelosa et al.10 In the former study, the Gromos96 43a1 force field was employed to examine different peptides spanning the 2F5 epitope, of which one (3f5 in ref 10) is the subject of this study. In contrast to this study, they found that the gp41 peptide does not adopt a stable structure in aqueous solution; rather, an equilibrium of interchanging globular structures is found in which hydrophobic packing is maximized. Turns and helices are also found to be members of this conformational ensemble. The presence of helices is consistent with our own results, although their simulation predicts comparable levels of 310- and π-helical content, thus underestimating 310-helix content relative to our own. It is likely that the discrepancies between simulations owe their origin to
Figure 4. Ramachandran plot at 844 K.
the differing force fields employed in each work: recent work23 has shown that the 43a1 version of Gromos yields significant quantities of π-helix content. A simpler conformational ensemble is suggested by the recent REMD simulations of Lapelosa et al.,10 in which an atomistic model of the peptide in conjunction with an implicit description of the solvent degrees of freedom indicates that the ELDKWAS peptide interconverts between R-helical and type-I β-turn
Conformational Plasticity structures. While our results indicate that the type-I β-turn is not a stable motif, it may be that the 7-mer considered by Lapelosa et al. has different structural propensities to the 13mer considered in our work, explaining the discrepancy. Additionally, the use of an implicit solvent model may produce a different free energy landscape from that obtained using an atomistic solvent model.24 Lastly, the simulations in ref 11 employ the optimized potential for liquid simulations all-atom force field; this model may predict different structural propensities to the CHARMM22 force field used in this work. Recalling that at higher temperatures (>800 K) our simulations exhibit an equilibrium of conformers not unlike those of ref 10, it would seem that the CHARMM force field acts to excessively stabilize helical structures at the expense of other secondary structure elements. A recent and thorough analysis25 suggests that classical force fields do overexpress helical content and that different force fields may differ widely in the regions of dihedral space sampled. This overstabilization of the helical structure in CHARMM appears, therefore, to introduce an “effective temperature shift”, whereby it is only at artificially high temperatures that an ensemble of secondary structure elements at the residue level is obtained. In summary, the simulation results indicate that the peptide exists in a conformational ensemble over the epitope region, with a mixture of R- and the rare 310-helices; the latter are confirmed as a persistent motif. Our simulations indicate that, outside the epitope region, the peptide is much more flexible and contains hydrogen bonding of side chains. Turn motifs are not found at ambient temperature, with analysis of the secondary structure propensities as a function of temperature indicating that they are only present at higher temperatures (T > 644 K). Comparison with the results of previous simulation data suggests that the CHARMM force field overstabilizes the helical structure. We note here that (see Supporting Information) these conclusions are not substantially altered by either changing water model or employing the CMAP26-28 correction to the CHARMM22 force field, although residue-specific propensities may alter. 4.2. Comparsion to Experiment. Various comparisons can now be drawn to previous experimental data. The NMR data are contradictory: the findings of Biron et al.1 suggest that the 310-helix is the dominant secondary structure with the R-helix appearing as a minority population, while Barbato et al.’s7 work does not indicate the presence of 310 helices, instead suggesting that an ensemble of conformers is present, including helices and turns. Our simulations indicate that the β-turn motif suggested by Barbato et al. is not stable, being readily annealed out. We find the 310-helix to be less stable than the R-helical motif for all empirical potentials. As discussed in the Supporting Information S1, however, there is considerable solvent and temperature sensitivity for certain residues, suggesting that buffer conditions in the experiment (which are not exactly matched in the simulations) may account for some of the difference in populations. It is only at elevated temperatures that our results are in qualitative agreement with NMR and UV resonance Raman measurements, which supplied evidence that the CHARMM force field overexpresses helical content.25 With regard to UV resonance Raman data,8 various amide bands can be taken as indirect reporters of secondary structure. Much of the analysis is based on the features of the Amide III band (combination of C-N stretch and N-H bending modes in the range spanning 1200-1350 cm-1) and the analysis of Mikhonin et al.,29 which gives a prescription by which the φ dihedral angle can be inferred from the spectra. The physical origins of the connection lie in the apparent sinusoidal variation29
J. Phys. Chem. B, Vol. 114, No. 23, 2010 7947 in the CR-H and N-H bending modes with ψ dihedral angle. By contrast, the Amide III bands vary only weakly with the φ backbone angle. The analysis is, however, complicated because it is necessary to separate the hydrogen bonding dependence of the Amide III bands from that on backbone conformation.29 In the spectroscopic data,8 a broad Amide III band is observed near 1257 cm-1. Whereas it is expected that a pure R-helical backbone conformation gives rise to a narrow Amide III band, here the spectroscopic observation is taken as evidence for multiple conformations. The 1254 cm-1 band represents a 9 cm-1 red shift relative to R-helical peptides and is assigned as the spectroscopic signature of the 310-helix. The authors point out that while no strong spectroscopic evidence exists for high R-helical populations some proportion may be consistent with the breadth of the Amide III bands. On the basis of the temperature dependence of the Amide III band and that of CR-H bending bands, it was noted that a similar behavior was observed for water-exposed pPII-type peptide and therefore that pPII may also be present. These authors also observe no decrease in helicity over the temperature range 1-30 °C in agreement with Biron.1 Other spectral features (e.g., the 1224 cm-1 band) indicate the presence of β-turn motifs. Also significant is the sub-band of the Amide III complex centered around 1293 cm-1. Again, following the data of Mikhonin et al.,29 this band is assigned to a dihedral angle φ ≈ 75° which corresponds to π-helix (i f i + 5) hydrogen bonding patterns. While we also observe some 310-helical content (i f i + 3 hydrogen bonds) in solution at 300 K, some of the evidence from our simulation results is at odds with the optical spectroscopy data; we find that turn motifs are not stable, and we also observe no evidence of π-helical conformations, the existence of which is inferred from the previous optical spectroscopic data. At ambient temperature, the energy landscape in the simulated system therefore appears simpler than spectroscopic data suggest; however, at elevated temperatures, the coexistence of several different conformers is characteristic of a flexible structure and suggests that the peptide possesses a rugged energy landscape. The roughness of the gp41 free energy landscape has been inferred by other authors,8 while Barbato et al. have posited that the conformational flexibility exhibited by the gp41 peptide is intimately linked to its biochemical functionality. This roughness suggests that the gp41 peptide exhibits a level of conformational plasticity, by which we mean that competing fold propensities may be present and which may be influenced by modifying the local microenvironment. Conformational plasticity has been proposed to be relevant to peptide and protein evolution in response to perturbations.30,31 It has also been suggested that conformational plasticity has a fundamental role in target protein recognition, as for example in the calmodulin protein,32-34 while several authors have examined structural plasticity in the context of fusion peptides.35-37 The presence of conformational plasticity in the gp41 peptide suggests that this may indeed be germane to understanding the mechanism underpinning the fusogenic process. The fact that no π-helix is observed in our simulations is consistent with the expectations38 that it is only stable experimentally in very exceptional cases. By contrast, we note that in some cases (e.g., ref 39 and references cited therein) the π-helix is favored over R for CHARMM22 force fields and has been observed in numerous simulated systems, such as the hydrophobic surfactant protein C in water (in which π f R conversion occurred after ≈300 ps). In other peptides known experimentally to form R-helical conformations, the simulated system exhibited transient appearances
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Figure 5. Far-UV CD spectra of 0.04 mM gp41659-671 peptide in different buffers at 5 °C in a 0.1 cm path length cuvette. Line: H2O pH 7.9. Dash: 20 mM sodium phosphate buffer pH 6.6. Dot and dash: 20 mM ammonium acetate buffer pH 7.7.
of π-helices. Sampling of π-helices typically is significantly reduced for the CHARMM22/CMAP force field. In the present case, however, π-helices are not observed, even without the correction, for either force field or the water model (see Supporting Information, S1). 5. Experimental Results At ambient temperature, the end residues of a helical peptide, which are not stabilized in the helical structure by hydrogen bonds, may be subject to pronounced “fraying”; this manifests itself as increased flexibility for these residues. Reducing the temperature may minimize this fraying, owing to restricted thermal motion, and thus one can expect that, for a peptide with helical propensity, a more helical structure will be obtained under these conditions.39 We thus investigate the CD spectra of the peptide at a temperature of 5 °C to ascertain as to whether any helical content is present. The far-UV CD spectra for gp41659-671 acquired at 5 °C in different buffers are shown in Figure 5. The far-UV CD spectrum in all the aqueous conditions tested (water, ammonium acetate buffer, sodium phosphate buffer) is typical of an ensemble of conformers in a flexible conformation. The lack of a positive band around 195 nm, together with the lack of bands at wavelengths higher than 200 nm, indicate the absence of any stable secondary structure elements. In particular, helical motifs typically exhibit two bands of negative ellipticity at 208 and 222 nm and a positive band at 195 nm, which are markedly absent from the spectra, thus allowing us to conclude that the peptide does not adopt a stable helical (R or 310) structure in solution. It should be pointed out that the intensity of R-helical CD increases with the chain length,40,41 and therefore the signal arising from only a few residues with backbone angles in the R-helical region of the Ramachandran plot may not be visible by CD. Turns can give variable signatures in CD, but the main feature of these elements of secondary structure is the presence of positive or negative bands at wavelengths above 200 nm. In particular, the CD spectra of type-I turns are of similar shape to that of the R-helix, while type-II turns give rise to signatures close in shape to that of the β-sheet, however, in both cases, with minima and maxima at different wavelengths. None of these signatures are observed in the far-UV CD spectra of the gp41659-671, indicating that the turn is not a stable secondary
Figure 6. Far-UV CD spectra of 0.04 mM peptide in 20 mM sodium phosphate buffer pH 6.6 over a range of temperatures.
structure element for this peptide. Finally, it should be noted that the presence of two Trp residues in the peptide sequence and their contribution in the far-UV region, in particular the ones around 225-230 nm, could render interpretation of spectral features around these wavelengths difficult.40,41 In general, two kinds of spectra are observed for unordered polypeptides. A first type of spectrum is very similar to the one usually assigned to the polyproline II (pPII) helix. This similarity has been used as a basis for the hypothesis that unfolded proteins possess considerable pPII helical content.42 A stable pPII helix is characterized by a negative peak around 200 nm and a weak positive band around 217 nm,13,42 where the position of the negative peak depends, in the case of the pPII helix, on the amino acid composition and is due to differences in transition energies between primary, secondary, and tertiary amines.42-44 A second type of unordered spectrum also shows a strong negative band at wavelengths below 200 nm but presents, at higher wavelengths, a negative shoulder and not a positive peak. The spectrum of the gp41659-671 peptide resembles this second kind of spectrum which is believed to reflect a more flexible conformation, with residues occupying the R-helix and/or β-sheet region of the Ramachandran plot,44 compared to the first type of unfolded spectrum that is pPII helix-like. The pPII conformation is stabilized at low temperature,13 and thus one means of detecting its presence is to measure CD spectra at different temperatures. We therefore examine the CD spectra as a function of temperature (Figure 6). The changes observed in the CD spectra upon increasing the temperature are typical of a very flexible structure (Figure 6). At high
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Figure 7. Changes in the far-UV CD spectra of 0.04 mM peptide in H2O as a function of percentage of TFE at 5 °C. Line: no TFE. Dash: 10% TFE. Dash and dots: 20% TFE. Dots: 30% TFE. The cuvette path length used is 0.2 cm.
temperatures, where the peptide chain conformation is expected to be irregular and disordered, only minor changes are observed in the spectra, which indicates that only a small change in the peptide conformation has occurred (Figure 6). Thus, the conformation at ambient temperature is compatible with a flexible conformational ensemble. This behavior is consistent with a rugged energy landscape characterized by shallow minima. The individual amino acid residues along the sequence could adopt, transiently, angles typical of secondary structure elements, or there could be an equilibrium between different conformers at a chain level. These conclusions are consistent with those reported by Barbato et al.7 Their work reported NMR structures which showed a high degree of flexibility (PDB code: 1mzi): this has been proposed to be important for the physiological function of this part of gp41. Our CD spectra are also similar. To investigate the effect of the microenvironment that the peptide could experience in the proximity of the cell membrane, TFE titrations were performed on the peptide solution. TFE is widely known to often induce secondary structure (mainly helical) in short peptides, and it is believed to mimic the microenvironment close to a membrane surface by lowering the solvent dielectric constant.14,45-47 The gp41659-671 peptide is located close to, and partially overlaps, the membrane proximal external region (MPER). It is believed that the MPER undergoes a conformational change upon interaction with the membrane surface. In the experiments carried out, the ensemble of peptide conformations changes upon addition of TFE in such a way that there is a shift in the conformer population toward higher percentages of conformers in helical structure. This is indicated by the red shift of the positive peak and especially by the shift of the negative peak from 199 to 205 nm with the appearance of a small shoulder at around 220 nm (Figure 7). Because of this sensitivity of the peptide conformation to solution conditions, as demonstrated by the TFE titrations, the disagreement between data from different laboratories can be due to factors that are difficult to control, such as the presence of impurities from the peptide synthesis or the presence of a small percentage of aggregates (preliminary DLS and SECHPLC experiments (data not shown) indicate the presence of high MW aggregates in the peptide solutions at high concentrations. We are further investigating these findings).
The 13-residue fragment of the human immunodeficiency HIV-1 surface glycoprotein (peptide gp41659-671) is special in several respects: (1) it comprises the only sequential epitope for one of the known antibodies of HIV-1, and (2) several recent experimental measurements (NMR and optical spectroscopies) have previously failed to establish a consistent picture of the conformational preference in aqueous solution with some indicating that it is a model system for the relatively rare 310helix and others suggesting that turn motifs are relevant for binding the human monoclonal antibody 2F5. Here we have combined atomistic replica exchange molecular dynamics simulations with circular dichroism (over a range of temperatures and solvent conditions) to develop a definitive structural model of this system. From the CD data, we find that conformational equilibrium exhibits considerable solvent sensitivity: in aqueous solutions, CD spectroscopic evidence points to a flexible ensemble of conformers with no identifiable secondary structure motif being dominant. Minor R helical populations are consistent with the experimental data though dissolution in water/TFE mixed solvent considerably enhances helical propensities. Replica exchange MD on the CHARMM potential surface was performed in which the initial configuration comprised two interleaved arrays corresponding to helical and turn motifs and where the system was fully denatured at the highest temperature. The simulated system exhibits coexisting R (dominant) and 310 (minor) folds. While the presence of these motifs is not excluded by the CD data, the simulation overestimates helical propensities. Some residue-specific sensitivity to the water model is also observed. The 310-helix does not emerge as a dominant motif from either simulation or experiment, and this peptide is therefore not a model system for this fold type. The balance of all available evidence is that the gp41659-671 peptide exhibits a number of features that have apparent physiological relvance, and it is therefore a useful model system in that respect. These include at least (1) conformational plasticity in which competing folding propensities are present and can be influenced by local microenvironment and (2) specific fold groups such as turn motifs which are identifiable at elevated temperatures in replica exchange molecular dynamics and potentially relevant in antibody binding and helices (the preferred motifs in the simulated system and promoted in membrane mimicking solvents which is particularly relevant as this is a membrane proximal peptide, and therefore the helical motif may well be physiologially significant). Supporting Information Available: Figures S1-S5 and Table 1. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Biron, Z.; Khare, S.; Samson, A.; Heyak, Y.; Naider, F.; Anglister, J. A Monomeric 310-Helix Is Formed in Water by a 13-Residue Peptide Representing the Neutralizing Determinant of HIV-1 on gp41. Biochemistry 2002, 41, 12687. (2) Salzwedel, K.; West, J.; Hunter, E. A Conserved Tryptophan-Rich Motif in the Membrane-Proximal Region of the Human Immunodeficiency Virus Type 1 gp41 Ectodomain Is Important for Env-Mediated Fusion and Virus Infectivity. J. Virol. 1999, 73, 2469–2480. (3) Joyce, J.; Hurni, W.; Bogusky, M.; Garsky, V.; Liang, X.; Citron, M.; Danzeisen, R.; Miller, M.; Shiver, J.; Keller, P. Enhancement of R-Helicity in the HIV-1 Inhibitory Peptide DP178 Leads to an Increased Affinity for Human Monoclonal Antibody 2F5 but Does Not Elicit Neutralizing Responses in Vitro. J. Biol. Chem. 2002, 277, 45811–45820. (4) Trkola, A.; Pomales, A.; Yuan, H.; Korber, B.; Maddon, P.; Allaway, G.; Kattinger, H.; Barbas, C.; Burton, D.; Ho, D.; Moore, J. Cross-
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clade neutralization of primary isolates of human immunodeficiency virus type 1 by human monoclonal antibodies and tetrameric CD4-IgG. J. Virol. 1995, 69, 6609–6617. (5) Zwick, M.; Jensen, R.; Church, S.; Wang, M.; Steigler, G.; Kunert, R.; Katinger, H.; Burton, D. Anti-Human Immunodeficiency Virus Type 1 (HIV-1) Antibodies 2F5 and 4E10 Require Surprisingly Few Crucial Residues in the Membrane-Proximal External Region of Glycoprotein gp41 To Neutralize HIV-1. J. Virol. 2005, 79, 1252–1261. (6) de Rosny, E.; Vassell, R.; Jiang, S.; Kunert, R.; Weiss, C. Binding of the 2F5Monoclonal Antibody to Native and Fusion-Intermediate Forms of Human Immunodeficiency Virus Type 1 gp41: Implications for FusionInducing Conformational Changes. J. Virol. 2004, 78, 2627. (7) Barbato, G.; Bianchi, E.; Ingallinella, P.; Hurni, W.; Miller, M. D.; Ciliberto, G.; Cortese, R.; Bazzo, R.; Shiver, J. W.; Pessi, A. Structural Analysis of the Epitope of the Anti-HIV Antibody 2F5 Sheds Light into Its Mechanism of Neutralization and HIV Fusion. J. Mol. Biol. 2003, 330, 1101. (8) Ahmed, Z.; Asher, S. UV Resonance Raman Investigation of a 310Helical Peptide Reveals a Rough Energy Landscape. Biochemistry 2006, 45, 9068–9073. (9) Martins do Canto, A. M. T.; Palace Carvalho, A. J.; Prates Ramalho, J. P.; Loura, L. M. S. T-20 and T-1249 HIV fusion inhibitors’ structure and conformation in solution: a molecular dynamics study. J. Pept. Sci. 2007, 14, 442–447. (10) Lapelosa, M.; Gallicchio, E.; Arnold, G. F.; Arnold, G. F.; Levy, R. M. In silico vaccine design based on molecular simulations of rhinovirus chimeras presenting HIV-1 gp41 epitopes. J. Mol. Biol. 2009, 385, 675– 691. (11) Schibli, D.; Montelero, R.; Vogel, H. The Membrane-Proximal Tryptophan-Rich Region of the HIV Glycoprotein, gp41, Forms a WellDefined Helix in Dodecylphosphocholine Micelles. Biochemistry 2001, 40, 9570. (12) Sanbonmatsu, K. Y.; Garcia, A. E. Structure of met-enkephalin in explicit aqueous solution using replica exchange molecular dynamics. Proteins: Struct., Funct., Genet. 2002, 46, 225–234. (13) Bochicchio, B.; Tamburro, A. Chirality 2002, 14, 782–792. (14) Siligardi, G.; Drake, A.; Mascagni, P.; Rowlands, D.; Brown, F.; Gibbons, W. Eur. J. Biochem. 1991, 199, 545–551. (15) MacKerell, A.; Bashford, D.; Bellott, M.; Dunbrack, R.; Evanseck, J.; Field, M.; Fischer, S.; Gao, J.; Guo, H.; Ha, S.; Joseph-McCarthy, D.; Kuchnir, L.; Kuczera, K.; Lau, F.; Mattos, C.; Michnick, S.; Ngo, T.; Nguyen, D.; Prodhom, B.; Reiher, W.; Roux, B.; Schlenkrich, M.; Smith, J.; Stote, R.; Straub, J.; Watanabe, M.; Wiorkiewicz-Kuczera, J.; Yin, D.; Karplus, M. All-Atom Empirical Potential for Molecular Modeling and Dynamics Studies of Proteins. J. Phys. Chem. B 1998, 102, 3586. (16) Tuckerman, M. E.; A Yarne, D.; Samuelson, S. O.; Hughes, A. L.; Martyna, G. J. Exploiting multiple levels of parallelism in Molecular Dynamics based calculations via modern techniques and software paradigms on distributed memory computers. Comput. Phys. Commun. 2000, 128, 333. (17) Martyna, G. J.; Klein, M. L.; Tuckerman, M. Nose´-Hoover chains - the canonical ensemble via continuous dynamics. J. Chem. Phys. 1992, 97, 2635. (18) Martyna, G. J.; Tuckerman, M. E.; Tobias, D. J.; Klein, M. L. Explicit reversible integrators for extended systems dynamics. Mol. Phys. 1996, 87, 1117. (19) Earl, D. J.; Deem, M. W. Parallel tempering: Theory, applications, and new perspectives. Phys. Chem. Chem. Phys. 2005, 23, 3910. (20) Coluzza, I.; Frenkel, D. Virtual-move parallel tempering. Chem. Phys. Chem 2005, 6, 1779. (21) Samuelson, S. O.; Tobias, D. J.; Martyna, G. J. Modern Computational Methodology Applied to the Simulation of Blocked Trialanine Peptide in Vacuo, Water Clusters and Bulk Water. J. Phys. Chem. B 1997, 101, 7592–7603. (22) Frishman, D.; Argos, P. Knowledge-based protein secondary structure assignment. Proteins: Struct., Funct., Genet. 1995, 23, 566–579. (23) Matthes, D.; de Groot, B. L. Secondary structure propensities in peptide folding simulations: a systematic comparison of molecular mechanics interaction schemes. Biophys. J. 2009, 97, 599–608.
Tulip et al. (24) Zhou, R.; Berne, B. J. Can a continuum solvent model reproduce the free energy landscape of a β -hairpin folding in water. Proc. Natl. Acad. Sci. 2002, 99, 12777–12782. (25) Best, R. B.; Buchete, N.-V.; Hummer, G. Are current molecular dynamics force fields too helical. Biophys. J. 2008, 95, L07–L09. (26) Mackerell, A. D., Jr.; Feig, M.; Brooks, C. L., III. Extending the treatment of backbone energetics in protein force fields: Limitations of gasphase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations. J. Comput. Chem. 2004, 25, 1400. (27) Mackerell, A., Jr. Empirical Force Fields for Biological Macromolecules: Overview and Issues. J. Comput. Chem. 2004, 25, 1583. (28) Mackerell, A., Jr.; Feig, M.; Brooks, C. L., III. Improved Treatment of the Protein Backbone in Empirical Force Fields. J. Am. Chem. Soc. 2004, 126, 698–699. (29) Mikhonin, A.; Bykov, S.; Myshakina, N.; Asher, S. Peptide Secondary Structure Folding Reaction Coordinate: Correlation between UV Raman Amide III Frequency, Ψ Ramachandran Angle, and Hydrogen Bonding. J. Phys. Chem. B 2006, 110, 1928. (30) Mun˜oz, V. Folding Plasticity. Nat. Struct. Biol. 2002, 9, 792–794. ¨ zbek, S. A biological cosmos of parallel universes: (31) Meier, S.; O does protein structural plasticity facilitate evolution. BioEssays 2007, 29, 1095–1104. (32) Ikura, M.; Ames, J. B. Genetic polymorphism and protein conformational plasticity in the calmodulin superfamily: two ways to promote multifunctionality. Proc. Natl. Acad. Sci. 2006, 103, 1159–1164. (33) Wilson, M. A.; Brunger, A. T. The 1.0 Å crystal structure of Ca2+bound calmodulin: an analysis of disorder and implications for functionally relevant plasticity. J. Mol. Biol. 2000, 301, 1237–1256. (34) Laine, E.; Yoneda, J. D.; Blondel, A.; Malliavin, T. E. The conformational plasticity of calmodulin upon calcium complexation gives a model of its interaction with the oedema factor of Bacillus anthracis. Proteins 2008, 71, 1813–1829. (35) Simon, S. A.; McIntosh, T. J. Pept.-Lipid Interact. 2002, 71, 1813– 1829. (36) Li, Y.; Tamm, L. K. Structure and plasticity of the human immunodeficiency virus gp41 fusion domain in lipd micelles and bilayers. Biophys. J. 2007, 93, 876–885. (37) Vaccaro, L.; Cross, K. J.; Kleinjung, J.; Straus, S. K.; Thomas, D. J.; Wharton, S. A.; Skehl, J. J.; Fraternali, F. Plasticity of influenza haemagglutinin fusion peptides and their interaction with lipid bilayers. Biophys. J. 2005, 88, 25–36. (38) Feig, M.; Mackerell, A., Jr.; Brooks, C. L., III. Force Field Influence on the Observation of π-Helical Protein Structures in Molecular Dynamics Simulations. J. Phys. Chem. B 2003, 107, 2831–2836. (39) Tatham, A. S.; Drake, A. F.; Shewry, P. R. Conformational studies of a synthetic peptide corresponding to the repeat motif of C hordein. Biophys. J. 1989, 259, 471–476. (40) Sreerama, N.; Woody, R. W. Computation and analysis of protein circular dichroism spectra. Numerical Methods, Part D, Methods in Enzymology; Academic Press, San Diego, 2004; Vol. 383, pp 383-351. (41) Sreerama, N.; Woody, R. W. Computation and analysis of protein circular dichroism spectra. Protein Sci. 1996, 5, 1108–1117. (42) Rucker, A.; Creamer, T. Protein Sci. 2002, 11, 980–985. (43) Bloemendal, M.; Johnson, W., Jr. Pharm. Biotechnol. 1995, 7, 65– 100. (44) Woody, R. W. Circular Dichroism and the Conformational Analysis of Biomolecules; Fasman, G. D., Ed.; Plenum Press: New York, 1993. (45) Waterhous, D.; Johnson, W., Jr. Biochemistry 1994, 33, 2121–2128. (46) Kentsis, A.; Sosnick, T. Trifluoroethanol Promotes Helix Formation by Destabilizing Backbone Exposure: Desolvation Rather than Native Hydrogen Bonding Defines the Kinetic Pathway of Dimeric Coiled Coil Folding. Biochemistry 1998, 37, 14613–14622. (47) Buck, M. Trifluorethanol and colleagues: cosolvents come of age. Recent studies with peptides and proteins. Q. ReV. Biophys. 1998, 31, 297–355.
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