Computational Studies of the Structural Stability of Rabbit Prion Protein Compared to Human and Mouse Prion Proteins

Prion diseases are invariably fatal and highly infectious neurodegenerative diseases affecting humans and animals. The neurodegenerative diseases such as Creutzfeldt-Jakob disease, variant Creutzfeldt-Jakob diseases, Gerstmann-Str$\ddot{a}$ussler-Scheinker syndrome, Fatal Familial Insomnia, Kuru in humans, scrapie in sheep, bovine spongiform encephalopathy (or ‘mad-cow’ disease) and chronic wasting disease in cattle belong to prion diseases. By now there have not been some effective therapeutic approaches to treat all these prion diseases. Dogs, rabbits and horses were reported to be resistant to prion diseases. By the end of year 2010 all the NMR structures of dog, rabbit and horse prion proteins (X-ray for rabbits too) had been finished to release into protein data bank. Thus, at this moment it is very worth studying the NMR and X-ray molecular structures of horse, dog and rabbit prion proteins to obtain insights into their immunity prion diseases. The author found that dog and horse prion proteins have stable molecular dynamical structures whether under neutral or low pH environments, but rabbit prion protein has stable molecular dynamical structures only under neutral pH environment. Under low pH environment, the stable $\alpha$-helical molecular structures of rabbit prion protein collapse into $\beta$-sheet structures. This article focuses the studies on rabbit prion protein (within its C-terminal NMR, Homology and X-ray molecular structured region RaPrP$^\text{C}$ (120-230)), compared with human and mouse prion proteins (HuPrP$^\text{C}$ (125-228) and MoPrP$^\text{C}$ (124-226) respectively). The author finds that some salt bridges contribute to the structural stability of rabbit prion protein under neutral pH environment.

💡 Research Summary

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This paper investigates the molecular basis of species‑specific resistance to prion diseases by comparing the structural stability of rabbit prion protein (RaPrP^C, residues 120‑230) with that of human (HuPrP^C, residues 125‑228) and mouse (MoPrP^C, residues 124‑226) prion proteins. All three species have had their NMR structures deposited in the Protein Data Bank (PDB) (rabbit 2FJ3, human 1QLX, mouse 1AG2), and the rabbit protein also possesses an X‑ray structure (3O79). The author performed extensive molecular dynamics (MD) simulations using AMBER 8 with explicit TIP3P water, exploring two pH conditions (neutral pH ≈ 7.4 and low pH ≈ 4.5) at three temperatures: 350 K (near physiological), 450 K (high‑temperature stress), and 500 K (extreme heat). Each simulation ran for up to 30 ns, and the trajectories were analyzed for root‑mean‑square deviation (RMSD), radius of gyration, secondary‑structure content, and the presence of salt bridges.

Key findings are as follows:

1. Temperature‑dependent behavior – At 500 K, rabbit PrP retains its α‑helical content under neutral pH but rapidly loses helices and forms β‑sheet structures under low pH. Human and mouse PrPs display a similar degree of destabilization at this temperature, but rabbit PrP shows a more pronounced loss of helices when the acidic environment removes critical salt bridges.

2. Effect of low pH – The low‑pH simulations reveal that specific salt bridges (notably Asp177‑Arg163 and Asp201‑Arg155) are stable in neutral conditions, contributing up to 20 % occupancy over the 30 ns trajectory. Acidic protonation disrupts these interactions, leading to a cascade of structural rearrangements: α‑helices unwind, β‑strands elongate, and the overall protein adopts a more expanded, less compact conformation.

3. Comparison with dog and horse PrPs – Previous work by the same group showed that dog and horse prion proteins maintain stable α‑helical structures across all pH and temperature conditions. In contrast, rabbit PrP is uniquely sensitive to acidic pH, suggesting that the salt‑bridge network in rabbit PrP is less redundant than in the other species.

4. Sequence and structural alignment – Multiple‑sequence alignment highlights residues that differ in rabbit PrP relative to the other species (e.g., S173, Q219, A224, L232, G228). These positions lie near the β2‑α2 loop, a region implicated in the conversion of PrP^C to the pathogenic PrP^Sc. The rabbit‑specific residues may affect local electrostatics and thus the propensity to form stabilizing salt bridges.

5. Homology and X‑ray model validation – Superposition of the homology model (6EPA) and the X‑ray structure (3O79) onto the NMR structure (2FJ3) yields RMSDs of 3.20 Å and 2.79 Å, respectively, confirming that the computationally built homology model is a reliable representation of the experimentally determined structures.

6. Biological implications – Prion disease pathology is driven by the conversion of α‑helical PrP^C into β‑sheet‑rich PrP^Sc. The present study suggests that rabbit PrP’s stability is contingent upon a set of pH‑sensitive salt bridges; when these are disrupted (as might occur in acidic intracellular compartments), the protein becomes prone to the α‑to‑β transition. Conversely, the more extensive and pH‑independent salt‑bridge network in dog and horse PrPs may underlie their observed resistance to prion infection.

7. Limitations and future directions – The simulations are limited to 30 ns and employ elevated temperatures that exceed physiological conditions, which may exaggerate unfolding events. Longer microsecond‑scale simulations, enhanced sampling techniques (e.g., metadynamics), and free‑energy calculations are needed to map the full conversion pathway. Experimental validation of key salt‑bridge mutants (e.g., D177N, R163A) would further substantiate the computational predictions.

In conclusion, the paper provides a detailed comparative MD analysis that links specific electrostatic interactions to the differential pH‑dependent stability of rabbit versus human and mouse prion proteins. The findings reinforce the concept that species‑specific sequence variations modulate the salt‑bridge network, thereby influencing the susceptibility or resistance to prion disease. This work lays a foundation for rational design of therapeutic strategies that could stabilize PrP^C by targeting critical salt bridges, and it highlights the value of high‑resolution structural data combined with advanced computational modeling in elucidating protein misfolding diseases.