The structural transformation comparison of the human Prion protein mutants V176G, E196A, and I215V by using molecular dynamics simulation

The point mutations in the gene coding of prion protein (PrP) originate human familial prion protein (HuPrP) diseases. Such diseases are caused by several amino acid mutations of HuPrP including V176G, I215V, and E196A located at the second, third native helix and in their loop, respectively. Determining the transition from cellular prion protein (PrPc) to pathogenic conformer (PrPSc) in the globular domain of HuPrP that results in pathogenic mutations is the key issue. The effects of mutation on monomeric PrP are detected in the absence of an unstructured N-terminal domain only. A MD simulation for each of these wild type mutants is performed to examine their structure in the aqueous media. The structural determinants are discerned to be different for wild-type HuPrP (125–228) variants compare to that of HuPrP mutations. These three mutations exhibiting diverse effects on the dynamical properties of PrP are attributed to the variations in the secondary structure, solvent accessible surface areas (SASAs), and salt bridges in the globular domain of HuPrP. High uctuations that are evidenced around residues of the C-terminus of the helix 1 for V176G cause Gerstmann-Straussler-Scheinker (GSS) syndrome. Conversely, the occurrence of uctuations around residues of helix 2, helix 3, and the loss of salt bridges in these regions for E196A and I215V mutants is responsible for Creutzfeldt-Jakob disease. Furthermore, small changes in the overall SASAs mutations strongly inuence the intermolecular interactions during the aggregation process. The comparative results in this study demonstrate that the three mutants undergo different pathogenic transformations.


Introduction
The neurodegenerative disease epidemic raises with the increase of life expectancy in the developed countries. It is an age-related disorder which affects both humans and animals. Prion diseases are a class of rare, fatal and progressive kind of neurodegenerative diseases including an extensive range of clinicopathological phenotypes (Lafon et al. 2018). They are distinctive infectious agents which are assembled from self-propagating multi-chain of misfolded host-encoded prion protein (PrP) ( It is acknowledged that the major reasons for these maladies are the posttranslational transformation of the ubiquitous cellular form of the prion protein (HuPrPc) to misfolded pathogenic isoform (HuPrPSc) (Wille and Requena 2018; Wulf et al. 2017). Furthermore, without exhibiting any covalent alterations, they eventually aggregate with a de ned structure (Dai et al. 2019;Vallabh et al. 2020). The quest for determining the mechanisms of Human (Hu) familial prion diseases caused by the mutations of prion protein is never-ending. The structural characteristics of PrPSc have not been entirely identi ed and the research is open to achieve the ultimate insight into its molecular mechanism. The main barrier in this way is the insoluble nature of PrPSc which prevents the use of high-resolution techniques for whole determination of its structure. Thus, a partial structure of PrPSc with a low resolution is available.
It is well-known that HuPrPSc structure is rich in β-strands whereas HuPrPc mainly consists of α-helix in secondary structure conformation. The structures obtained from NMR reveal a C-terminal globular domain from residue 125 to 228 (human numbering) and an N-terminal exible disordered tail (Poggiolini et al. 2013). The globular domain is composed of three α-helices forming the residues 144-154 helix 1, 173-194 helix 2, and 200-228 helix 3 as well as a very short anti-parallel β-sheet with residues 128-131 b1 and 161-164 b2. Also, a disulphide bond (Cys179-Cys214) connects helices 2 and 3.  Figure 1 shows the methionine and valine polymorphisms that are preserved for these mutants at codon-129.

Molecular Dynamics Simulation
The GROMACS 5.0.7 software package and all-hydrogen function GROMOS96 were employed to run MD simulations over 100 ns (Parvizpour et al. 2019). The simulations were done in the aqueous solution using a water molecule cubic box with a vector size of 7.08 nm for all prion systems. The protonation states of the ionizable residues are adjusted to maintain the neutral pH conditions regarding their pKas, using Na ions as counter ions. Periodic boundary conditions in the NPT ensemble are used at a xed pressure (P = 1 bar) and temperature (T= 300 K). The Ewald particle mesh (PME) is used to treat longrange electrostatic interactions. are twisted together from 3 to 100 ns as illustrated in Figure 2B. A relatively higher average RMSD response (100 ns) for I215V mutant than wild-type mutant structures is observed. This is ascribed to the higher structural instability of I215V compare to WT PrP structure. This higher response arises from the strong conformational change in their molecular structure. RMSD value for WT structure is found to be higher than that of the V176G mutant as depicted in Figure 2A. Thus, these deviations in the mutant structure caused by the disease display unusual features (Simpson et al. 2013).

RMSFs of C α Atom
The RMSFs display signi cant initial uctuations. Irrespective of runs, the structures exhibit random uctuations in the residues range of 3-29. All mutants consist of a loop, β-strand, and helix 1 structures. The minimum uctuations are evidenced for helix 2 and helix 3 regions associated with a single disulphide bridge. Conversely, the maximum uctuations are appeared in the helix 1 region (residues [19][20][21][22][23][24][25][26][27][28][29] for V176G and other mutants. However, the RMSF values as shown in Figure 3A are observed to be very stable for V176G in helix 2 (residues 48-69) and helix 3 (residues 75-103). Mutants E196A and I215V corresponding to helix 2 and helix 3 regions are detected to be more exible (less stable) than WT as illustrated in Figures 3B and 3C. In all cases the higher RMSFs for HuPrP mutants compare to that of WT PrP indicates their lower rigidity. Furthermore, the amino acid substitutions at helix 2, helix 3, and within their loop introduce more exibility due to long range interactions. Truly, these interactions are responsible for the uctuations in mutant structures and in uence the stability of PrP. Mutations might have some local impact on the protein interactions which are required for oligomerization into brillar species (Behmard et al. 2011).

Percentage of Secondary Structure
The secondary structural elements of HuPrP mutants and WT as displayed in Figure 4 are appeared to be largely preserved in all mutated systems over 100 ns timescale. In comparison to WT Figure 4A, the structural contents of helix 2 in V176G Figure 4B show a shift from helix to turn the structures in helix 3. Conversely, helix 2 of E196A Figure 4C and helix 3 of I215V Figure 4D  The most important features of protein misfolding and aggregation are described by hydrophobicity and SASA parameters. Estimations of these quantities are essential to determine the stability of secondary and tertiary structural elements of PrPs (van der Kamp and Daggett 2010). (Table 1) displays the computed values of hydrophobicity and SASA for HuPrP mutants. The hydrophobicity for each mutant V176G, E196A, and I215V reveals an increase compared to WT. However, the value of SASA for both E196A and I215V is increased and for V176G is decreased in comparison to WT. The electrostatic potential surfaces of all mutants are depicted in Figure 6. All mutated regions exhibit more color changes compared to that of WT. The electrostatic potential redistributions may in uence the intermolecular recognition and interactions between PrPSc and PrPc or the aggregation of PrPSc (Guo et al. 2012b). Our MD simulation results on the dynamical behavior of the globular domain of WT and the HuPrP mutants (V176G, E196A, and I215V) rea rm the earlier observations. It is demonstrated that different amino acid substitutions produce some subtle effects on the structural features of PrP. Although, helix 1 reveals higher uctuations compare to WT for V176G but both helix 2 and 3 remain very stable. However, mutants E196A and I215V show the opposite behavior compare to V176G. The uctuations grow up in helix 2 and 3 but remain more stable in helix 1. The contents of β-sheet do not increase in mutated PrPSc as expected. The disruption of a speci c SB network present in WT and mutants in all disease linked mutations consequently increase the exibility of helix 1 for V176G and helix 2 and 3 for E196A and I215V. The residues involved in the SBs are highly conserved and their absence is ascribed to the pathogenic behaviors (Adrover et al. 2010). The formation of PrPSc is assigned to the disruption of native SBs and transfer of some charged groups in a low permittivity environment.

Discussion
It is further observed that the alterations in the charge state of a mutant protein tend to form aggregates and the aggregation propensity of a polypeptide chain is inversely correlated with its net charge. Thus, point mutations strongly affect the kinetics of amyloid formation. The core of PrPSc amyloid is observed to be in favor of a low dielectric and hydrophobic environment (Adrover et al. 2010; Guest et al. 2010). Much of these destabilizing mutations are found to be located in the central hydrophobic core of PrP (Guo et al. 2012b). A region can be de ned as interacting residues with purely hydrophobic side chains that form the core of the globular domain. We assert that the hydrophobic core is important for the stability of the globular domain of PrP.

Conclusion
The change in human prion protein in residues located within sequences of HuPrP caused by mutation is simulated. Structural variations in WT PrP and disease related mutations caused by V176G and E196A (carrying valine) and I215V (carrying Methionin) at codon 129 are compared to determine the impacts of mutation on monomeric PrP. The MD simulation of 100 ns performed for wild type and each of these mutants in the globular domain is found to be fairly conserved between WT and mutants. The changes in the secondary structures and local uctuations are very small upon mutations. Three mutations are found to have diverse effects on the dynamical properties of PrP causing variations in the secondary structure, SASAs, and salt bridges in the globular domain of HuPrP. Furthermore, high uctuations occur around residues of the C-terminus of the helix 1 for V176G compare to other mutants such as E196A and I215V display somewhat unusual constellation of molecular dynamic features. Our analyses reveal the conformational uctuations in the mutant's structure. The mechanism of the transformation of PrPc to PrPSc that results in prion diseases is established. The domain in the C-terminal end of the protein plays a signi cant role in transforming PrPc to PrPSc and results in most of the pathogenic mutations within this part of the protein is focused without considering unstructured N-terminal.