Unique Structural Characteristics of the Rabbit Prion Protein*

Rabbits are one of the few mammalian species that appear to be resistant to transmissible spongiform encephalopathies due to the structural characteristics of the rabbit prion protein (RaPrPC) itself. Here, we determined the solution structures of the recombinant protein RaPrPC-(91–228) and its S173N variant and detected the backbone dynamics of their structured C-terminal domains-(121–228). In contrast to many other mammalian PrPCs, loop 165–172, which connects β-sheet-2 and α-helix-2, is well-defined in RaPrPC. For the first time, order parameters S2 are obtained for residues in this loop region, indicating that loop 165–172 of RaPrPC is highly ordered. Compared with the wild-type RaPrPC, less hydrogen bonds form in the S173N variant. The NMR dynamics analysis reveals a distinct increase in the structural flexibility of loop 165–172 and helix-3 after the S173N substitution, implying that the S173N substitution disturbs the long range interaction of loop 165–172 with helix-3, which further leads to a marked decrease in the global conformational stability. Significantly, RaPrPC possesses a unique charge distribution, carrying a continuous area of positive charges on the surface, which is distinguished from other PrPCs. The S173N substitution causes visible changes of the charge distribution around the recognition sites for the hypothetical protein X. Our results suggest that the ordered loop 165–172 and its interaction with helix-3, together with the unique distribution of surface electrostatic potential, significantly contribute to the unique structural characteristics of RaPrPC.

form encephalopathy in cattle, and Creutzfeldt-Jakob disease in humans, are an unusual group of fatal neurodegenerative disorders that can be sporadic, inherited, or acquired. The infectious agent has been identified uniquely as the scrapie prion protein (PrP Sc ), a pathogenic isoform of the host-encoded cellular prion protein (PrP C ) (1,2). PrP C and PrP Sc seem to possess the same covalent structure but differ substantially in conformation. PrP C is monomeric and soluble, sensitive to proteolysis with proteinase K, whereas PrP Sc is highly insoluble and readily forms proteinase-resistant aggregates. Both circular dichroism (CD) spectra and Fourier transform infrared (FTIR) spectra show that PrP C is predominantly ␣-helical, whereas PrP Sc possesses a considerable amount of ␤-sheet content (3)(4)(5).
PrP C is mostly expressed in the central nervous system. It is a highly conserved cell surface glycoprotein, attached to the outer leaflet of the cell membrane via a glycosylphosphatidylinositol anchor at its C terminus. Although the physiological function of PrP C remains unknown, its high affinity for copper(II) indicates that it may act as a copper transport protein or a superoxide dismutase (6,7). Previous studies have indicated that PrP C may be involved in pathways related to cell adhesion, synaptic integrity, and cell signaling (8,9).
The conformational conversion of the prion protein, from PrP C to PrP Sc , has a crucial role in the pathogenesis of TSEs. Thus, knowledge of the three-dimensional structure of the prion protein is of great importance to understand the conformational conversion. Solution structures of nonglycosolated PrP C s across a number of mammalian species, including mice, Syrian hamsters, humans, cattle, sheep, tammar wallabies, horses, etc., have been determined using NMR spectroscopy (10 -16). Mature PrP C consists of a single polypeptide chain of ϳ210 amino acids (residues . The N terminus (residues 23-120) is flexible and disordered, with a highly conserved octa-repeating sequence PHGGGWGQ between residues 60 and 91. The C terminus (residues 121-231) is a globular structured domain encompassing three ␣-helices and two short antiparallel ␤-strands, with a disulfide bond bridging helices 2 and 3. Loop 165-172 and helix-3, so-called conformational markers, are located on the surface of the structured C-terminal domain and reflect the differences among species (17).
Rabbits are one of the few mammalian species that appear to be resistant to TSE agents. So far, no signs of TSE diseases have been observed in rabbits after inoculating them with the Creutzfeldt-Jakob disease, kuru, or scrapie agents isolated from either mice or sheep (18). Both rabbit PrP C (RaPrP C ) and chimeric rabbit-mouse PrP C constructs are not converted to the proteinase-resistant form in scrapie-infected mouse neuroblas-toma cells (Sc ϩ -MNB cells), which accumulate mouse PrP Sc (mPrP Sc ) (19). These experiments suggest that the inability of the conformational conversion for RaPrP C and the resistance to the TSE infection for rabbits is most likely due to the structural characteristics of the RaPrP C protein itself. Thus, interpretation of the three-dimensional structure of RaPrP C would reveal the particular properties of RaPrP C distinguished from other PrP C s. In addition, sequence alignment shows that there are 22 different amino acid residues between RaPrP C and mouse PrP C (mPrP C ). The mPrP C variants with multiple amino acid residues substituted by the corresponding residues in RaPrP C (N99G, L108M, N173S, or V214I) are inhibited to convert to the abnormal form (19). These experimental observations imply that the specific amino acid residues at pivotal spots may somehow determine the structural characteristics of PrP C . Therefore, a detailed understanding of tertiary structural differences between RaPrP C and mPrP C , caused by the specific amino acid residues, could provide an essential insight into the molecular mechanism by which TSEs develop.
In the present work, we utilized multidimensional heteronuclear NMR techniques to determine the solution structures of the recombinant protein RaPrP C -(91-228) and its S173N variant. Moreover, we used CD spectroscopy to assess their conformational stability upon urea-induced denaturation. In addition, we performed 15 N relaxation measurements to detect the backbone dynamics of their globular structured C-terminal domains-(121-228). Our results reveal the unique structural characteristics of RaPrP C and offer invaluable hints to understand the molecular mechanism of the conformational conversion for prion proteins.

Plasmid Construction, Protein Expression, and Purification-
The amplified gene fragment coding for RaPrP C -(91-228) was inserted into the vector pET30a via NdeI and XhoI restriction sites. A single point mutant on this plasmid containing a serine 173 to asparagine substitution (S173N) was constructed via the site-directed mutagenesis PCR. Two other truncations, RaPrP C -(121-228) and its S173N variant, were constructed using the vector pGBTNH via BamHI and XhoI restriction sites. The expression of recombinant proteins was carried out in the E. coli BL21(DE3) strain at 37°C. Unlabeled proteins were prepared in LB medium. Uniformly 15 N/ 13 C-labeled and 15 N-labeled proteins were prepared by culturing cells in M9 minimal medium with 15 N-labeled ammonium chloride in the presence or absence of 13 C-labeled glucose, respectively. The on-column purification and refolding of recombinant proteins was performed as described previously (20), with the only modification by adding thrombin protease to remove the GB1 tag contained in the truncated proteins. Protein samples were desalted into Buffer F (20 mM NaOAc, 0.02% NaN 3 , pH 4.5) and concentrated to 0.25 ml using Ultrafree-15 Centrifugal Filter Biomax Devices (Millipore). The protein purity was checked by polyacrylamide gel electrophoresis under denaturing condition, and the protein concentration was measured by the BCA assay (Sigma). NMR samples at a concentration of 1 mM with 10% D 2 O were used for structure determination and dynamics analysis.
NMR Spectroscopy-All NMR measurements were carried out at 25°C on a Varian Unity Inova 600 spectrometer equipped with three RF channels and a triple-resonance pulsed-field gradient probe. The spectra were processed with the program NMRPipe (21) and analyzed by the software Sparky (T. D. Goddard and D. G. Kneller, University of California, San Francisco). The three-dimensional heteronuclear NMR spectra, including HNCA, HN(CO)CA, HNCACB, CBCA(CO)NH, HBHA(CO)NH, HNCO, and HN(CA)CO, were collected to obtain the sequence-specific backbone resonance assignments. The three-dimensional spectra H(CC)(CO)NHTOCSY, (H)CC(CO)NHTOCSY, HCCH-TOCSY, CCHTOCSY, and 15 N-edited TOCSY-heteronuclear single quantum coherence were recorded for the side chain resonance assignments. Nearly complete backbone and side chain resonance assignments for RaPrP C -(91-228) (22) and the S173N variant have been obtained. The three-dimensional 15 Nand 13 C-edited NOE spectroscopy-heteronuclear single quantum coherence experiments with a 100-ms mixing time were performed to confirm the resonance assignments and generate 1 H-1 H distance restraints for structure calculation. The hydrogen-deuterium exchange experiments were conducted to obtain hydrogen bond restraints.
Structure Determination-The three-dimensional structures of RaPrP C -(91-228) and the S173N variant were calculated using NOE-derived distance restraints, in combination with dihedral angle restraints and hydrogen bond restraints by ARIA/CNS software (23,24). A family of 200 structures was calculated according to the simulated annealing protocol. Fifteen structures of the lowest energy were selected, which exhibited no NOE violation Ͼ0.3 Å and no dihedral angle violation Ͼ5°. The final 15 structures were assessed by the PROCHECK program (25). The structural statistics are presented in Table 1. The ribbon and surface graphs were displayed using the software MolMol (26) or PyMOL (kindly provided by Professor W. L. DeLano). The atomic coordinates of RaPrP C -(91-228) and the S173N variant have been deposited into the Protein Data Bank (PDB codes 2FJ3 and 2JOH). The chemical shift data are available at the Biological Magnetic Resonance Data Bank (accession nos. 7142 and 16328). 15 N Relaxation Measurements-All 15 N relaxation data were acquired at 25°C on a Varian Unity Inova 600 spectrometer. The standard pulse sequences with minimal water suppression (27) were used to record the spectra of relaxation times T 1 , T 2 , and { 1 H}-15 N heteronuclear NOE. In the direct ( 1 H) dimension, the carrier frequency was set on the water resonance with a spectral width of 10,000 Hz. In the indirect ( 15 N) dimension, the spectral width was 1420 Hz. A recycle delay of 2 s was used. T 1 was measured using a series of spectra recorded with 10 relaxation delays: 10 15 N NOE enhancements were calculated as the ratio of peak heights in spectra recorded with or without proton saturation. The saturated spectra were acquired with a 2-s relaxation delay followed by a 3-s period of proton saturation. The spectra recorded in the absence of proton saturation employed a relaxation delay of 5 s. The S.E. were determined from two data sets.
Reduced Spectral Density Mapping-The spectral density mapping approach can be used to describe the internal motions of N-H bonds without any assumptions about a specific molecular model. The heteronuclear relaxation parameters can be obtained from a weighted sum of the spectral density function J() at five specific frequencies: 0, N , H , H Ϫ N , and H ϩ N . At high frequencies, however, the values of J(0.87 H ), J( H ϩ N ), and J( H Ϫ N ) can be assumed to be approximately equal in the case of 15 N relaxation (28 -30). Thus, it is possible to map the spectral density function only using relaxation rates R 1 , R 2 , and heteronuclear NOEs. The reduced spectral density values can be expressed as follows, 0 is the permeability of the free space, h is the Planck's constant, ␥ H and ␥ N are the gyromagnetic ratios of 1 H and 15 N, respectively, r NH is the N-H bond length, H and N are the Lamor frequencies of 1 H and 15 N, respectively, and ⌬ is the chemical shift anisotropy for 15 N with ⌬ ϭ ʈ Ϫ Ќ ϭ Ϫ160 ppm. We used the notebook provided by Spyracopoulos (31) to execute the calculation.
Model-free Analysis-The model-free formalism is often used to describe internal motions of a protein. The spectral density functions are modeled differently depending on whether the rotational diffusion tensor is either isotropic or anisotropic. In the formalism of Lipari and Szabo (32,33), the spectral density function is given by the following, where Ϫ1 ϭ m Ϫ1 ϩ e Ϫ1 , S 2 is the generalized order parameter that specifies the degree of spatial restriction of the N-H bond, m is the correlation time for overall tumbling, and e is the correlation time for internal motion. Clore et al. (34) expanded this formalism to account for internal motion on two distinct time scales, which differ by at least one order of magnitude. An exchange term, R ex , is also incorporated to account for slower conformational exchange processes which affect transverse relaxation times. The rotational diffusion tensors of RaPrP C -(121-228) and the S173N variant were evaluated for a selected subset of spins with trimmed R 2 /R 1 ratios (35) as well as NOE values Ͼ0.6. We used the program FASTModelFree (36) to perform model selection and model-free analysis.
Circular Dichroism-All CD spectra were recorded on a Jasco J-810 spectropolarimeter at 25°C. The reported spectra were an average of three consecutive scans and corrected for blank.
Far-UV CD spectra were collected in the wavelength range of 200 -250 nm using a 0.1-cm path length quartz cuvette. For denaturation experiments, a high concentration stock of folded protein was diluted into Buffer F with or without urea, reaching a final protein concentration of 0.2 mg⅐ml Ϫ1 and to a desired urea concentration. For renaturation, the high concentration urea in the stock of unfolded protein was diluted by added denaturant-free protein in Buffer F. The urea-induced unfolding transitions for secondary structure were analyzed by the mean residue ellipticity () at 222 nm, assuming a two-state mechanism and a dependence of on denaturant concentration (37, 38), where [] O is an estimate of the free energy of unfolding extrapolated to zero concentration of denaturant, m N and m U are coefficients of the dependence of ellipticity on denaturant concentration at native or unfolded state, m is a measure of the dependence of ⌬G N3U on denaturant concentration, R is the gas constant, and T is the absolute temperature.
Near-UV CD spectra were collected in the region of 260 -350 nm using a 1-cm path length quartz cuvette. Stock solutions contained 1 mg⅐ml Ϫ1 protein with or without urea. Fresh mixtures of the stocks at the desired urea concentration were measured. The mean residue ellipticity at 268 nm was used for analysis.

RESULTS
Solution Structures-The three-dimensional structure of RaPrP C -(91-228) comprises two short antiparallel ␤-strands (S1, residues 128 -130; S2, 160 -162) and three ␣-helices (H1, 144 -153; H2, 173-186; H3, 199 -227) (Fig. 1, A and B), similar to those of other mammalian prion proteins. Helices-2 and -3 are further stabilized by the disulfide bond between Cys 178 and Cys 213 . The region 91-126 is highly disordered, owing to a lack of NOEs. Loop 165-172 and helix-3, designated as conformational markers (17), form a solvent-accessible contiguous epitope at the C terminus of the prion protein. This area has been suggested to be the recognition sites for a hypothetical chaperone, protein X, which would moderate the conversion of PrP C into the pathogenic PrP Sc (39,40). Noticeably, loop 165-172 can be assigned completely in RaPrP C (supplemental Fig. S1) by long range NOEs from the residues at the end of helix-3. mPrP C with the N173S substitution was not able to convert to the abnormal form (19). Thus, it is expected that the S173N substitution would cause structural change more or less for RaPrP C . To evaluate the structural change, we determined the solution structure of the S173N variant of RaPrP C -(91-228) (Fig. 1, C and D). Both three-dimensional structures are identical overall but with some local differences. The orientation of helix-1 in the variant is divergent to that of the wild-type, with a deviation angle of ϳ24°. The end of helix-3 displays an irregular bend for either the wild-type or the variant; however, the bend is a bit sharper for the latter. Residues 191-193 may form a 3 10 helix in the variant.
Surface Charge Distribution-It has been suggested that the distribution of electrostatic potential on the surface of the prion protein is related to the transmission barrier of TSEs (13,14,41,42). We compared the surface charge distributions among RaPrP C and other mammalian PrP C s including human PrP C , mPrP C , and bovine PrP C (Fig. 2). Three-dimensional structures of these PrP C s were determined in sodium acetate buffer at pH 4.5 (10,12,13). Amazingly, the distribution of electrostatic potential for RaPrP C , with a large area of continuous positive charge on the surface ( Fig. 2A), is distinguished significantly from those for other PrP C s (Fig. 2). The area of continuous positive charge of RaPrP C roughly covers residues 125-135, 150 -160, and 180 -190. Electrostatic interactions could distinctly influence on the binding specificity and affinity of a protein with substrates. This unique charge distribution is most likely to have a profound effect on the ability of RaPrP C to interact with other molecules.
The S173N substitution causes marked changes in the surface charge distribution for RaPrP C . RaPrP C -(91-228) displays a neutral charge distribution around the position of residue 173, whereas the S173N variant carries negative charge in the same position (Fig. 3, A and B). Surface charge changes are observed not only in the right substituted position but also at other sites such as residue Gln 219 , from a negative charge in the wild-type to a neutral charge in the variant (Fig. 3, C and D). A discontinuous epitope of residues 167, 171, 214, and 218 has been suggested to be involved in the binding of PrP C with the putative protein X (40). Misfolding and propagation of the prion protein mediated by protein X is likely to occur if the surface-restricted electrostatic potential would not work as a barrier for intermolecular interactions.
Hydrogen-bonding Networks-Hydrogen bonds usually have important roles in stabilizing tertiary structures. We compared the hydrogen bonding networks between the wild-type RaPrP C -(91-228) and the S173N variant. As a criterion, hydrogen bonds are thought to be stable if they exist in at least 9 structures among the 15 lowest energy structures. In all, 55 hydrogen bonds are detected in the wild-type; however, only 47 are found in the variant, implying that the S173N substitution decreases the structural stability. A set of hydrogen bonds, which make significant contribution to maintain the tertiary structure for either the wild-type or the variant, are listed in Table 2. The distribution and number of hydrogen bonds in RaPrP C -(91-228) is dramatically different from that of the S173N variant (Table 2). Therefore, it could be expected that  OCTOBER 8, 2010 • VOLUME 285 • NUMBER 41 the structural change in the S173N variant is more or less readily to be induced by other chaperones.

Unique Rabbit Prion Protein
Conformational Stability-To evaluate the effect of the S173N substitution on the conformational stability, we measured the urea-induced unfolding transitions of both RaPrP C -(121-228) and the S173N variant using CD spectroscopy (Fig.  4). Far-UV CD spectra show the change of secondary structure with the increase of urea concentration (Fig. 4, A and B). Due to irreversible denaturation for either the wild-type or the variant (supplemental Fig. S2, A and B), we determined the apparent   O and the midpoint denaturant concentration C m are lessened after the S173N substitution ( Table 3), suggesting that the conformational stability of the S173N variant is decreased compared with that of the wild-type. The coefficient m, which is linked to the solvent accessible surface area and hydrophobic interactions (43)(44)(45)(46), also is significantly altered with the S173N exchange ( Table 3), indicative of distinct difference in hydrophobic interactions between the wild-type and the variant.
Near-UV CD spectra reflect the change of tertiary structure against urea concentration (Fig. 4, C and D). Both the wild-type and the S173N variant show the minimal values at 268 nm; thus, the mean residue ellipticity () at this wavelength is selected for analysis. The trends of 268 are coincident with theoretical sigmoid curves, similar to those of 222 . The midpoint C m values are ϳ5.3 M for the wild-type and 4.9 M for the S173N variant, implying a lower conformational stability after the S173N substitution. The C m values in the near-UV CD spectra are much smaller than the corresponding values in the far-UV CD spectra ( Fig. 4 and Table 3), which suggests that the change of tertiary structure is prior to that of secondary structure for the two proteins upon urea denaturation. For a higher urea concentration, however, the change of secondary and tertiary structures occurs simultaneously.
Relaxation Rates R 1 and R 2 and { 1 H}-15 N Heteronuclear NOE of RaPrP C -(121-228)-A complete description of the tertiary structure of a protein requires a well understanding of how the structure changes with time. The measurement of 15 N spin relaxation parameters for N-H bond vectors provides valuable information about internal dynamics of proteins on both picoto nanosecond and micro-to millisecond time scales (47,48). Recent work has demonstrated that changes in motions can influence protein functions even if conformations are the same (49). To gain an insight into the backbone dynamics of RaPrP C -(121-228) in solution, we measured 15 N longitudinal (T 1 ), transverse (T 2 ) relaxation times and { 1 H}-15 N heteronuclear NOEs. Totally, 97 N-H resonances were assigned and used in the dynamics analysis for RaPrP C -(121-228). We utilized peak height to represent peak intensity so that the disturbance of There are no significant differences in low J(0.87 H ) values for most residues in helices-1, -2, and -3, which indicates that these three helices are subjected to little internal motions on fast picosecond time scale.
Order Parameters S 2 of RaPrP C -(121-228)-The model-free formalism usually is used to analyze internal motions of a protein (32)(33)(34). The D ʈ /D Ќ ratio of the rotational diffusion tensor of RaPrP C -(121-228) is calculated to be 1.31 Ϯ 0.01 using the R2R1_diffusion program (kindly provided by Professor A. G. Palmer III), suggesting that the axially symmetric model is suitable for the data fitting. We determined the order parameter S 2 for each residue in RaPrP C -(121-228) (Fig. 7A). The order parameter quantitatively describes the amplitude of the internal motion on a nanosecond time scale. Residues with S 2 Ͻ0.7 are mostly observed in three flexible loop fragments including residues 121-126, 138 -141, and 190 -198. However, loop 165-172, which connects ␤-sheet-2 and ␣-helix-2, exhibits large S 2 values Ͼ0.85, indicating a highly ordered loop. Residues in secondary structure elements have relatively higher S 2 values, typically with values Ͼ0.8, indicative of rigid regions with restricted internal motions. The mean S 2 values for residues in helices-1, -2, and -3 are 0.93, 0.94, and 0.85, respectively. The S 2 values are mapped onto the tertiary structure of RaPrP C and shown in Fig. 7C.
Backbone Dynamics of the S173N Variant-To investigate whether the S173N substitution would influence the internal motions of the RaPrP C molecule, we analyzed the backbone dynamics of the structured C-terminal domain-(121-228) of the S173N variant. The overall distributions of relaxation parameters (Fig. 5B) and the reduced spectral density functions (Fig. 6B) for the S173N variant are similar to those for the wildtype. The value differences of the reduced spectral densities for two local regions, including loop 165-172 and the half-end of helix-3, between the wild-type and the S173N variant are shown in Fig. 6C. Increased internal flexibility usually causes a decrease of the J( N ) value for small proteins (50,54). Greater subnanosecond flexibility can be also reflected in smaller J(0) values (50). Unlike insignificant changes in ⌬J(0.87 H ), most of the residues in loop 165-172 and at the half-end of helix-3 exhibit positive values in ⌬J( N ) and ⌬J(0) (Fig. 6C), implicating more internal mobility of the two regions in the S173N variant. Thus, the S173N substitution potentially disturbs the interaction between loop 165-172 and helix-3. Furthermore, as the  7B) and mapped them onto the tertiary structure of the S173N variant (Fig. 7D). Unexpectedly, the end of helix-3 in the variant exhibits S 2 values Ͻ0.8 (Fig. 7B), dramatically smaller than those of the corresponding residues in the wild-type (Fig. 7), suggesting that the S173N substitution significantly increases  OCTOBER 8, 2010 • VOLUME 285 • NUMBER 41 structural flexibility. Usually, the local region of high flexibility is associated with a lower energy barrier to structural rearrangement. Therefore, the conformational rigidity of RaPrP C may allow this protein to resist to the induction of other molecules and successfully escape from the conformational conversion.

DISCUSSION
Rabbits are among the few mammalian species that are capable of surviving with the infection of TSE agents due to some unique structural characteristics of the RaPrP C molecule itself (19). In this present work, we determined the solution structures of the recombinant protein RaPrP C -(91-228) and its S173N variant and detected the backbone dynamics of their globular structured C-terminal domains-(121-228). Kurt Wuthrich and co-workers (13,55) have proved that the threedimensional structure of the labeled recombinant bovine PrP C is identical to that of the unlabeled natural glycoprotein isolated from calf brains, which provides solid evidence that isotopic labeling used in bio-NMR does not effect the conformation of the protein. Our results also show that the conformations of labeled or unlabeled RaPrP C s are essentially identical (supplemental Fig. S3, A and B). Thus, the tertiary structure presented here represents the true conformation of the RaPrP C molecule. Previous studies have revealed that the homologous loop 165-172 is related to prion diseases (56,57). NMR resonances in loop 165-172 are unobservable for many mammalian PrP C s such as mPrP C , human PrP C , or bovine PrP C (10,12,13). Only a few mammalian PrP C s, including elk PrP C , tammar wallaby PrP C , and horse PrP C (15,16,58) have not missed resonances. Loop 165-172 in RaPrP C can be well assigned by long range NOEs from the residues at the end of helix-3. Recently, the model-free approach has been employed successfully to elucidate the backbone dynamics for mPrP C -(121-230) (59). Nevertheless, order parameters S 2 for the corresponding loop in mPrP C are not available due to invisible backbone resonances. We have demonstrated that the well defined loop 165-172 in RaPrP C is highly ordered, supported by the S 2 values measured for the first time, providing an important insight into the species barrier of the infection of TSEs. The S173N exchange does not influence the overall scaffold of the RaPrP C molecule; however, the urea-induced transition experiments have illustrated a decrease in global conformational stability after the substitution. Molecular dynamics simulations do not find the difference of PrP C stability among species (60) but confirm the structural stability of the wild-type RaPrP C compared with the variants and show that the salt bridge between Asp 177 and Arg 163 makes a great contribution (61). Such a salt bridge, however, is not observed in our work possibly owing to the low pH condition. Our results have indicated that the decreased conformational stability after the Ser 173 substitution is relevant to distinct changes of hydrogen bonding networks and hydrophobic interactions. In particular, the S173N substitution causes an increase in structural flexibility of loop 165-172 and the end of helix-3, implying an alteration in the interaction of loop 165-172 with helix-3. The interaction between these two regions is implicated to have a crucial role in stabilization of the PrP C structure (62). Previous studies demonstrate that helix-1 is rather stable against environmental perturbations and unlikely to be involved in the initial steps of the pathogenic conformational change (63,64). Recent reports show that helix-2 and helix-3 in the structured C-terminal domain of the PrP C protein are most likely to undergo a transition to ␤-structure (65-67). Therefore, the highly ordered loop 165-172 and its interaction with helix-3 may form a huge barrier to prevent the C-terminal domain of RaPrP C from the conformational conversion. Christen et al. (15) have inferred that the interaction between these two conformational markers could act as regulators of the functional specificity of PrP C . The study reported by Hill and co-workers (68) suggests that the rabbitspecific residues surrounding the C-terminal glycosylphosphatidylinositol anchor may interfere with a PrP Sc interaction site.
It is unlikely for PrP C to undergo a spontaneous transition without any induction of the chaperone in the complicated cellular environment. Previous studies have suggested that PrP Sc , protein X, and a specific nucleic acid could bind to PrP C and induce conformational changes (2, 39, 40, 69 -71). The surface charge distributions around binding sites usually have important roles in determining the binding specificities of proteins with substrates (72,73). One single residue change can lead to significant influences on the surface charge distribution of a protein such as the S173N variant of RaPrP C described herein and the E200K variant of human PrP C reported by Zhang et al. FIGURE 7. Shown are the order parameters S 2 for RaPrP C -(121-228) (A) and the S173N variant (B). The regular secondary structure elements are indicated at the top. S 2 values are mapped onto the tertiary structures of RaPrP C -(121-228) (C) and the S173N variant (D). Red, S 2 Ͻ 0.6; orange, 0.6 Յ S 2 Ͻ 0.7; yellow, 0.7 Յ S 2 Ͻ 0.8; green, 0.8 Յ S 2 Ͻ 0.9; blue, 0.9 Յ S 2 Ͻ 1.0; and gray, S 2 unavailable due to the absence of data or failure in data fitting. The ribbon graphs are generated using PyMOL. OCTOBER 8, 2010 • VOLUME 285 • NUMBER 41 (41). The distribution of electrostatic potential for RaPrP C , with a large area of continuous positive charge on the surface, is dramatically different from those of other PrP C s, which would make significant contribution to protect RaPrP C from the conformational conversion. PrP Sc has been identified as the infectious agent of TSEs. Transgenetic studies indicate that the formation of PrP Sc requires the PrP C substrate to bind to the PrP Sc product at an intermediate stage of the conformational conversion process (69). PrP Sc could recognize the surface of PrP C containing fragments 90 -144 and 180 -205 (2). The area of continuous positive charge on the surface of RaPrP C roughly covers residues 125-135, 150 -160, and 180 -190. Thus, the interaction of PrP C with PrP Sc is likely to be inhibited due to the exclusive distribution of electrostatic potential on the surface of RaPrP C . In addition, much evidence supports the hypothesis that the conformational conversion of PrP C into PrP Sc is a key molecular event for the pathogenesis of TSEs (74 -77). Both DNA and RNA may stimulate the misfolding process of prions (70,71). Recent studies have reported that the DNA-PrP C interaction is mediated mainly through the globular structured domain of PrP C , with a recruitment of residues in the N-terminal unstructured loop 91-120 (78,79). Electrostatic contacts are one of the most important factors for DNA or RNA recognition by proteins. The unique surface charge distribution of RaPrP C , especially the continuous positive-charged region contained in the globular C-terminal domain, may significantly influence the electrostatic interaction of RaPrP C with the specific nucleic acid. The distinct nucleic acid-RaPrP C interaction potentially leads to the inability of RaPrP C to undergo the conformational transition. The partially exposed residue His 186 has been demonstrated to be able to disrupt the surrounding hydrophobic interactions upon protonation at acidic pH, resulting in destabilization of the C-terminal half of helix-2 (59). His 186 also is involved in the copper(II) binding (80,81), which may cause a decrease in the structural stability of helix-2. On the other hand, PrP Sc could recognize a surface area of PrP C containing fragments 90 -144 and 180 -205 (2). Destabilization of PrP C is required for the interaction of PrP C with PrP Sc in vitro (82)(83)(84). In addition, our NMR dynamics analysis shows that His 186 undergoes significant internal motions although it is located in a secondary structure element (helix-2). Thus, the residue His 186 is expected to act as a trigger for the initial binding of PrP C to PrP Sc . However, only this step may be not sufficient for the conformational conversion of prions as RaPrP C is not yet converted to the proteinase-resistant form in Sc ϩ -MNB cells (19). We thus suggest that the highly ordered loop 165-172 and its interaction with helix-3, together with the unique distribution of surface electrostatic potential, significantly contribute to the unique structural characteristics of RaPrP C . Our results will be helpful for better understanding the underlying molecular mechanism of the conformational conversion for prion proteins.