Solution Structure and Dynamics of the I214V Mutant of the Rabbit Prion Protein

Background: The conformational conversion of the host-derived cellular prion protein (PrP C) into the disease-associated scrapie isoform (PrP Sc) is responsible for the pathogenesis of transmissible spongiform encephalopathies (TSEs). Various single-point mutations in PrP C s could cause structural changes and thereby distinctly influence the conformational conversion. Elucidation of the differences between the wild-type rabbit PrP C (RaPrP C) and various mutants would be of great help to understand the ability of RaPrP C to be resistant to TSE agents. Methodology/Principal Findings: We determined the solution structure of the I214V mutant of RaPrP C (91–228) and detected the backbone dynamics of its structured C-terminal domain (121–228). The I214V mutant displays a visible shift of surface charge distribution that may have a potential effect on the binding specificity and affinity with other chaperones. The number of hydrogen bonds declines dramatically. Urea-induced transition experiments reveal an obvious decrease in the conformational stability. Furthermore, the NMR dynamics analysis discloses a significant increase in the backbone flexibility on the pico- to nanosecond time scale, indicative of lower energy barrier for structural rearrangement. Conclusions/Significance: Our results suggest that both the surface charge distribution and the intrinsic backbone flexibility greatly contribute to species barriers for the transmission of TSEs, and thereby provide valuable hints fo

Solution Structure and Dynamics of the I214V Mutant of the Rabbit Prion Protein

Background: The conformational conversion of the host-derived cellular prion protein (PrPC) into the disease-associated scrapie isoform (PrPSc) is responsible for the pathogenesis of transmissible spongiform encephalopathies (TSEs). Various single-point mutations in PrP(C)s could cause structural changes and thereby distinctly influence the conformational conversion. Elucidation of the differences between the wild-type rabbit PrPC (RaPrPC) and various mutants would be of great help to understand the ability of RaPrPC to be resistant to TSE agents. Methodology/Principal Findings: We determined the solution structure of the I214V mutant of RaPrPC (91-228) and detected the backbone dynamics of its structured C-terminal domain (121-228). The I214V mutant displays a visible shift of surface charge distribution that may have a potential effect on the binding specificity and affinity with other chaperones. The number of hydrogen bonds declines dramatically. Urea-induced transition experiments reveal an obvious decrease in the conformational stability. Furthermore, the NMR dynamics analysis discloses a significant increase in the backbone flexibility on the pico- to nanosecond time scale, indicative of lower energy barrier for structural rearrangement. Conclusions/Significance: Our results suggest that both the surface charge distribution and the intrinsic backbone flexibility greatly contribute to species barriers for the transmission of TSEs, and thereby provide valuable hints for understanding the inability of the conformational conversion for RaPrPCNational Natural Science Foundation of China [30730026, 30570352]; National Science & Technology, China [2009ZX09301-001

Apparent thermodynamic parameters for the equilibrium unfolding of RaPrP^C(121–228) and the I214V mutant at 25°C.

<p>Note: is an estimate of the free energy in the absence of denaturant, the parameter represents the cooperativity of the unfolding transition, and is the concentration of urea at the midpoint of unfolding. The determined parameters for the wild-type <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013273#pone.0013273-Wen1" target="_blank">[30]</a> are listed here to facilitate comparison.</p

Relaxation rates R₁, R₂ and {¹H}-¹⁵N heteronuclear NOEs of the I214V mutant of RaPrP^C(121–228).

<p>Regular secondary structure elements are indicated on the top. The relaxation constants and the experimental errors were extracted by a single exponential curve fitting of the peak heights using Sparky (T. D. Goddard and D. G. Kneller, University of California, San Francisco).</p

Modelfree analysis.

<p>(A) Order parameters S² of the I214V mutant of RaPrP^C(121–228). Regular secondary structure elements are indicated on the top. The program Fastmodelfree <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013273#pone.0013273-Cole1" target="_blank">[63]</a> was used to perform the calculation. Unavailable S² values for a few residues are due to the absence of data or failure in the data fitting. (B) Differences in S² values between the wild-type and the mutant. The difference is calculated according to the equation: ΔS² = S²_mutant−S²_wild-type. The absence of ΔS² values for residues result from unavailable S² values for either the wild-type or the mutant. (C) ΔS² values are mapped onto the tertiary structures of the I214V mutant: blue for ΔS²≥0, red for ΔS²<0, and grey for ΔS² unavailable. This ribbon diagram is generated by PyMol (kindly provided by Prof. DeLano WL).</p

Urea-induced transitions of the I214V mutant of RaPrP^C(121–228) characterized by CD spectroscopy.

<p>(A) The folded protein in the absence of urea (solid line) compared with the unfolded protein in the presence of 9 M urea (dashed line). (B) Mean residue ellipticity measured at 222 nm (θ₂₂₂) in the presence of urea of different concentration. θ₂₂₂ in denaturation is indicated by solid squares (▪), while in renaturation by open circles (○). The solid line presents the fitting curve on the basis of a two-state mechanism.</p

Solution structure of the I214V mutant of RaPrP^C(91–228).

<p>(A) Diagram of 15 lowest-energy conformations. (B) Ribbon cartoon showing the secondary structure elements of the mean structure. (C) Sausage diagram showing the superposition of structures of the wild-type (grey) and the mutant (pink). The structure was determined in acetate buffer at pH 4.5. The diagrams are generated using MolMol <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013273#pone.0013273-Koradi1" target="_blank">[61]</a>.</p

Reduced spectral density functions analysis.

<p>(A) Spectral densities of the I214V mutant of RaPrP^C(121–228). (B) Differences of spectral densities between the wild-type and the I214V mutant. The difference is calculated as follows: Δ<i>J</i>(ω) = <i>J</i>(ω)_mutant−<i>J</i>(ω)_wild-type. Regular secondary structure elements are indicated on the top. The notebook provided by Spyracopoulos <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013273#pone.0013273-Spyracopoulos1" target="_blank">[62]</a> was used to execute the calculation.</p