Atomic insights into the stability of the electrostatic network and hydrophobic core in the prion protein

Abstract

Destabilization of a prion protein (PrP) induces a conformational change that alters a normal prion protein (PrPC) to an abnormal prion protein (PrPSC). The structural stability of PrP is dramatically destabilized by unfavorable external environmental conditions and mutations in the protein. Environmental pH is a critical determinant of misfolding and aggregation of PrP, which leads to fatal neurodegenerative diseases. Because protonation of H187 and mutation of the hydrophobic core on the H2-H3 bundle are strongly linked to a change in PrP stability, I examined its charged residues R156, E196, and D202 around H187 and hydrophobic residues V176, V180, T183, V210, I215, and Y218. Interestingly, there are reports on mutants, such as V176G, T183A, H187R, E196A, D202N, I215V, and Y218N, which cause genetic prion diseases. First, I focused on the mechanism by which an acidic pH and mutants disrupt this electrostatic network and how this broken network destabilizes the PrP structure. Towards this objective, I performed a temperature-based replica-exchange molecular dynamics (T-REMD) simulation using a cumulative 252μs simulation time. I measured the distance between amino acids comprising four salt bridges (R156–E196/D202 and H187–E196/D202). Our results showed that the spatial configuration of the electrostatic network was significantly altered by an acidic pH and mutations. The structural alteration in the electrostatic network increased the RMSF value around the first helix (H1). Thus, the structural stability of H1 anchored to the H2–H3 bundle was decreased, which induced separation of R156 from the electrostatic network. Analysis of the anchoring energy also showed that the two salt-bridges (R156-E196/D202) are critical for PrP stability. Second, I focused on the hydrophobic interaction. Not only electrostatic interaction but also hydrophobic interaction is the main driving force for protein folding, critically affecting the stability and solubility of the protein. To examine the importance of the hydrophobic core in the PrP, I chose six amino acids (V176, V180, T183, V210, I215, and Y218) that form the hydrophobic core at the middle of the H2-H3 bundle. A few pathological mutants of these amino acids have been reported, such as V176G, V180I, T183A, V210I, I215V, and Y218N. I revealed how these mutations affect the hydrophobic core and thermostability of PrP. Towards this, I used a temperature-based replica-exchange molecular dynamics (T-REMD) simulation for extensive ensemble sampling. From the T-REMD ensemble, I calculated the protein folding free energy difference between wild-type and mutant PrP using the thermodynamic integration (TI) method. Our results showed that the mutants V176G, T183A, I215V, and Y218N decreased PrP stability. At the atomic level, I examined the change [valine-valine to valine-isoleucine (and vice versa)] in pair-wise hydrophobic interactions, which is induced by mutation V180I, V210I (I215V) at the 180th–210th (176th–215th) pair. Additionally, I investigated the importance of the π-stacking between Y218 and F175.