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2009, 113, 12853–12856 Published on Web 09/02/2009
Regulating the Conformation of Prion Protein through Ligand Binding Norifumi Yamamoto and Kazuo Kuwata* DiVision of Prion Research, Center for Emerging Infectious Diseases, Gifu UniVersity, 1-1 Yanagido, Gifu 501-1194, Japan, and CREST Project, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan ReceiVed: June 15, 2009; ReVised Manuscript ReceiVed: August 12, 2009
Although some antiprion compounds have been shown to interfere with the pathological conversion of prion protein into a misfolded isoform, the actual mechanism has not been elucidated. In this study, we compared different conformations of prion protein with and without ligand binding, based on molecular dynamics simulations, to clarify the role of a typical antiprion compound termed GN8. In our approach, urea-driven unfolding simulations were employed to assay whether or not GN8 prevents denaturation of prion protein. We found that urea mediates partial unfolding at helix B of the prion protein, suggesting a transition into the intermediate states of the pathological conversion. However, GN8 efficiently suppressed local fluctuations by binding to flexible spots on helix B and prevented its urea-induced denaturation. We conclude that GN8 inhibits pathological conversion by suppressing the level of the intermediate. This is the first evidence supporting the chemical chaperone hypothesis, which states that GN8 acts as a chaperone to stabilize the normal form of the prion protein. Our basic principle constitutes a promising strategy for a dynamics-based drug design of therapeutic compounds, particularly for prion diseases and other diseases related to protein misfolding. Prion diseases are associated with conformational conversion of a normal cellular form of prion protein (PrPC) into an alternatively folded scrapie isoform (PrPSc).1 According to the most widely accepted protein-only hypothesis, PrPSc is an infectious agent, which is believed to self-perpetuate by a mechanism involving its binding to PrPC followed by the conversion of PrPC to PrPSc.2-4 The structural detail of PrPSc remains unresolved, and the mechanism of PrPC to PrPSc conversion is not completely understood. Moreover, little information is available about the infection mechanism of prion diseases; however, the experimental finding implying the intrinsic instability of PrPC is a focus for research. For example, a high-pressure nuclear magnetic resonance (NMR) study showed that the residues in helices B and C of PrPC undergo significant pressure-dependent chemical shifts.5 At the corresponding regions, slow fluctuations were observed on a time scale of microseconds to milliseconds.6 These results indicate that a partially unfolded state of PrPC, yielding metastable transient conformations, would be induced in response to given physiochemical perturbations. The actual in vivo conditions necessary for the formation of PrPSc are unknown; however, in vitro experiments have shown that destabilization of PrPC using chemical denaturants favors the formation of a conformational intermediate (PrP*) in the PrPC to PrPSc conversion.7 When the intrinsic instability of PrPC facilitates the conformational rearrangements occurring between PrPC and PrP*, even under normal physiological conditions, metastable PrP* would coexist with the native PrPC. Although present at low levels, PrP* is considered to be a candidate for * To whom correspondence should be addressed. Tel: +81-58-230-6145. Fax: +81-58-230-6144. E-mail:
[email protected].
10.1021/jp905572w CCC: $40.75
active intermediate species that is directly recruited into the pathological PrPSc. Recently, some antiprion compounds that specifically bind with the intrinsically unstable spots on helix B in PrPC have been discovered.8,9 The main compound GN8, N,N′-(methylenedi-4,1-phenylene)bis[2-(1-pyrrolidinyl)acetamide], was found to significantly inhibit PrPSc production in vitro.8 Moreover, in prion-infected mice, even after the appearance of clinical signs in the control group, the GN8-treated group showed prolonged survival.8 The possible antiprion mechanism of GN8 was predicted to be as follows: GN8 interacts with the hot spots at helix B, acting as a chemical chaperone, and exclusively interferes with the conformational conversion of PrPC to PrPSc by suppressing the intermediate PrP* levels.8 In this study, we provide the first evidence supporting the above-mentioned chemical chaperone hypothesis. To clarify the role of antiprion compounds, we compared different conformations of PrPC with and without GN8 binding, based on molecular dynamics (MD) simulations. In our approach, urea-driven unfolding simulations were employed to assay whether or not GN8 prevents the formation of PrP*. Furthermore, to better understand the antiprion mechanism from the viewpoint of molecular dynamics, we analyzed characteristic changes in conformational fluctuations through GN8 binding. All MD simulations were performed using the Gromacs 4.0.4 package10 with the Gromos96 43a2 force field11 in the NpT ensemble, where constant temperature and pressure were maintained at 300 K and 1 atm. The SPC/E water model12 and Smith’s urea model13 were employed to describe the solvents. According to the protein-only hypothesis, the fragment containing residues 90-231 of PrP, generated by amino-terminal truncation through digestion with proteinase K, is considered 2009 American Chemical Society
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Letters
Figure 1. Binding structure of the PrP-GN8 complex. A snapshot was taken from molecular dynamics steps performed in water. GN8 is represented by the “ball and stick” model. Three GN8-binding residues, Tyr155, Asn159, and Glu196, are drawn as “licorice” bonds. The image was created using VMD.24
Figure 2. Urea-induced partially unfolded structure of PrP. A snapshot was taken 80 ns after a denaturing molecular dynamics simulation in 6 M urea solution. Six residues in the denaturant susceptible site (residues 188-193) are represented by the “licorice” bonds. The image was created using VMD.24
to be the minimal infectious unit that retains prion infectivity and polymerizes into amyloid.4 Moreover, a previous experimental study showed that formation of the unfolding intermediate is independent of segments 90-120 and revealed an intrinsic property of the C-terminal domain of PrP(121-231).14 Therefore, in this study, instead of using the full-length prion protein, we used truncated coordinates of the C-terminal half domain of residues 124-226 in mouse PrPC (PDB code: 1AG2),15 which might include the minimal requirement for PrP* formation. The modeled structure of PrPC consists of three R-helix domains, termed helix A (HA; residues 143-156), helix B (HB; residues 171-193), and helix C (HC; residues 199-223), and two β-sheet domains, termed sheet 1 (S1; residues 128-131) and sheet 2 (S2; residues 160-163). Starting with the NMR structure of mouse PrPC,15 we first used a docking method to identify the most plausible initial structure of the PrP-GN8 complex. The initial coordination of the complex was produced using the ICM-Pro 3.0 program,16 by allowing complete flexibility of the side-chains of the receptor, where putative hydrogen bonds between GN8 and Asn159 and between GN8 and Glu196 were presumed to be situated. To refine the PrP-GN8 structure, a 100 ns MD simulation with no constraints was performed with water solvent. During the simulation, the complex structure underwent conformational rearrangements, whereas the essential intermolecular hydrogen bonds between GN8 and PrPC remained well preserved. The change in the radius of gyration and principal moments of inertia of the PrP-GN8 structure indicated that the GN8-binding complex is equilibrated after 20 ns in the simulation. The 20 ns equilibration phase was eliminated from the beginning of the 100 ns trajectory, and the 80 ns production phase was used for analyses of the PrP-GN8 complex simulated in water. To examine the urea-driven denaturing dynamics of the PrP-GN8 system, an 80 ns production run was performed, where the initial coordinate for the PrP-GN8 complex was obtained from an equilibrated water simulation. First, we analyzed the binding structure of the PrP-GN8 complex, simulated at the standard canonical condition in water. Figure 1 shows a snapshot taken from a MD trajectory within the production phase. As shown in this figure, GN8 binds to
PrPC by cross-linking distant residues, Asn159 in the HA-S2 loop and Glu196 in the HB-HC loop, by hydrogen bonds. The GN8-binding sites in the simulated structure agreed with the characteristic NMR chemical shift changes upon ligand binding.8 A recent quantum chemistry calculation of the PrP-GN8 complex revealed that the central diphenylmethane scaffold also contributes to the binding affinity by van der Waals interaction,17 for example, PrPC-GN8 binding with Tyr155, which is consistent with the NMR chemical shift changes of residues 155 and 157.8 We should note that GN8 binding causes disruption of the salt bridge between Arg156 at the C-terminus of HA and Glu196 in the HB-HC loop, which might play an important role in the conformational stability of PrPC. In fact, mutation of E196K causes rapidly progressive dementia and ataxia.18,19 In PrP-GN8, alternative intermolecular interactions between PrPC and GN8 would compensate for the lack of the original salt bridge, and the rearrangement of interactions could affect the ability to misfold. Next, we investigated the chemical denaturing of PrPC in an aqueous 6 M urea solution. Note that the midpoints of the ureainduced unfolding transition were reported to be 5.72 M for mPrP(121-231)14 and 5.88 M for mPrP(23-231).20 Figure 2 shows a typical example of denatured structures obtained from a trajectory after a simulation period of 80 ns. Figure 3 shows the ratios of the helix and sheet contents per residue during the productive phase of MD simulations in urea, where the secondary structure was assigned using the DSSP program.21 This figure also shows the changes in content ratios after denaturing, which indicates the differences in average helix and sheet contents between the two structures of PrP simulated in water and urea solutions, respectively. As shown in Figure 3a, when compared to the structure of PrPC in water, it is evident that the R-helices are markedly less well preserved in the simulations in urea. Major urea-induced changes in secondary structure were observed in the following parts: the C-terminus of HA (residues 154-156), the N-terminus of HB (residues 172-176), the C-terminus of HB (residues 188-193), and the C-terminus of HC (residues 221-223). These urea-induced
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Figure 3. Distribution of the secondary structure per residue for (a) PrP and (b) PrP-GN8. In each figure, the upper panel shows averaged content ratios of R-helix and β-sheet conformations per residue through the product phase of a urea-driven denaturing simulation. The lower panel shows variations in helix and sheet content ratios of the denatured PrP structure in aqueous urea solution from the equilibrated PrP structure simulated in water. In each figure, secondary structure elements determined on the basis of the NMR structure15 are shown in the middle.
conformational changes agree, in both distribution and amplitude, with the experimental results observed in the guanidinium solution.7 The C-terminus of HB (residues 188-193), which shows prominent denaturant weakness, is known as a threonine-rich region including the TVTTTT sequence. Intriguingly, such stretches are frequently observed in the context of β-strand conformations.22 Recently, Yamaguchi et al. revealed experimentally that the HB-derived peptide truncated from PrPC has a high propensity for β-strands and tends to form β-sheet-rich amyloid-like fibrils.23 The authors concluded that once the native structure is destabilized under conditions where the region of HB is accessible, then the prion protein will adopt non-native conformations with enhanced β-sheet contents instead of PrPC.23 It is expected that partial unfolding of the C-terminus of HB in PrPC would exert its effect on the initial stage of the pathological conversion to PrPSc that is believed to be a β-sheet-rich structure.
J. Phys. Chem. B, Vol. 113, No. 39, 2009 12855 Thus, the resulting urea-driven partially unfolded state could be regarded as the intermediate PrP* in the conversion pathway of PrPC to PrPSc. After GN8 binding, even in the 6 M urea solution, most of the protein moiety retains the secondary structure, except for the C-terminus of HC (see Figure 3b). Thus, the computational evidence presented here completely supports the concept that GN8 acts as a chemical chaperone to stabilize normal conformation. Here, we confirmed that GN8 binds to PrPC with the same binding mode that occurs in water during the ureadenaturing simulation. The loop motif in protein is generally flexible. In fact, a large degree of fluctuation within the HB-HC loop in PrPC was indicated by previous experimental studies.5,6 As shown in Figure 3a, the partial unfolding at the N- and C-termini of the R-helices can be attributed to the fluctuating motions of the loop domain. GN8 binds to PrPC by cross-linking two loop domains, the HA-HB and HB-HC loops. Thus, regulating conformational fluctuation through ligand binding to unstable spots is believed to be the key antiprion mechanism of GN8. To better understand the effect of ligand binding on structural flexibility, we investigated the root-mean-squared deviation (rmsd) of CR-CR distances as an indicator of conformational fluctuation. Figure 4 shows the time-averaged rmsd values of the simulations for PrP and PrP-GN8 in a given environment. In the case of PrP in water, as shown in the left upper half of Figure 4a, while most inter-residue motions inside of each helix domain were expected to be minor fluctuations, several residues at the terminus of the helix exhibited relatively large rmsd values. This may be because of the fluctuations of the adjacent flexible loop domains. Other characteristics were the interhelix fluctuations between a pair of the C-termini of HA and HB and a pair of the C- and N-termini of HB and HC. These helices interact through the salt bridge among charged residues of Arg156 in HA, Glu196 in the HB-HC loop, and Asp202 in HC. Thus, the large conformational fluctuation might be attributed to a Coulomb interaction via the salt bridge. In the urea solution, as shown in the lower right half of Figure 4a, the characteristics of the conformational fluctuations are expected to be quite similar, except for the pairs of residues adjacent to the denatured moiety at the C-terminus of HB. Intriguingly, as shown in Figure 2a, the fluctuating regions shown in Figure 4a undergo denaturation in the urea solution. These results indicate that PrPC is fragile at the hot spots of intrinsic conformational flexibility, even in water, which might induce local destabilization and promote partial unfolding of PrPC. Thus, it is expected that the flexibility of the C-terminal region of HB can facilitate the initial stage of the partial unfolding process of PrPC to PrP*. After GN8 binding, as shown in Figure 4b, the degree of fluctuation was considerably reduced overall. In particular, the intrahelix fluctuations at the N- and C-termini of HB and the interhelix fluctuations between the C-terminus of HB and the Cand N-termini of HA and HC, respectively, were effectively suppressed by ligand binding. Thus, it is expected that GN8 binding, even in the urea solution, protects the hot spots and prevents partial folding of PrPC, followed by PrP* formation. In summary, we found that GN8 binding can efficiently suppress local fluctuations and prevent unfolding of PrPC under denaturation. The resulting urea-induced partially unfolded state can be regarded as a putative PrP* intermediate in the PrPC to PrPSc conversion. Therefore, we conclude that GN8 inhibits the pathological conformational conversion from PrPC to PrPSc by suppressing the PrP* intermediate levels.
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Letters Acknowledgment. This work was supported by the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO) and by a Grant-in-Aid for Young Scientists (B) from the Ministry of Education, Culture, Sports, Science and Technology (No. 20750008), Japan. Supporting Information Available: Simulation details. This material is available free of charge via the Internet at http:// pubs.acs.org. References and Notes
Figure 4. The rmsd of CR-CR distances as a function of residue number for (a) PrP and (b) PrP-GN8. In each figure, the upper left half of the diagram represents rmsd values during a simulation in water, and the lower right half shows the values during a denaturing simulation in urea. A pair of two residues of PrP binding with GN8, Asn159, and Glu196 is represented by a filled circle.
The basic principle presented in this study constitutes a promising strategy of dynamics-based drug design, aided by in silico antimisfolding assays of therapeutic compounds, especially for prion diseases and for other diseases related to protein misfolding.
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