Folding of the Ig-Like Domain of the Dengue Virus Envelope Protein Analyzed by High-Hydrostatic-Pressure NMR at a Residue-Level Resolution

Abstract

Dengue fever is a mosquito-borne endemic disease in tropical and subtropical regions, causing a significant public health problem in Southeast Asia. Domain III (ED3) of the viral envelope protein contains the two dominant putative epitopes and part of the heparin sulfate receptor binding region that drives the dengue virus (DENV)’s fusion with the host cell. Here, we used high-hydrostatic-pressure nuclear magnetic resonance (HHP-NMR) to obtain residue-specific information on the folding process of domain III from serotype 4 dengue virus (DEN4-ED3), which adopts the classical three-dimensional (3D) ß-sandwich structure known as the Ig-like fold. Interestingly, the folding pathway of DEN4-ED3 shares similarities with that of the Titin I27 module, which also adopts an Ig-like fold, but is functionally unrelated to ED3. For both proteins, the unfolding process starts by the disruption of the N- and C-terminal strands on one edge of the ß-sandwich, yielding a folding intermediate stable over a substantial pressure range (from 600 to 1000 bar). In contrast to this similarity, pressure-jump kinetics indicated that the folding transition state is considerably more hydrated in DEN4-ED3 than in Titin I27.

Combining High-Pressure Perturbation with NMR Spectroscopy for a Structural and Dynamical Characterization of Protein Folding Pathways

“…Even though characterizing transition states in protein folding constitutes an essential step in the puzzle [48], the relations between the protein sequences, their 3D structures, and the structure (at least the hydration state) of their TSE are not yet well understood. Thus, the HP-NMR comparative study of the folding of Titin I27 module and DEN4-ED3 domain from the viral envelope of the dengue virus, two proteins with unrelated sequences but sharing a common Ig-like fold, shows similar folding intermediates but very different TSE, the transition state of Titin I27 being considerably less hydrated than the one of DEN4-ED3 [49]. Such analysis relies on the measurement of kinetic parameters after perturbation (P-jump) of the thermodynamic equilibrium between the folded and unfolded conformers of the protein at a given pressure, yielding the rates of folding and unfolding at atmospheric pressure.…”

Loosening of Side-Chain Packing Associated with Perturbations in Peripheral Dynamics Induced by the D76N Mutation of β2-Microglobulin Revealed by Pressure-NMR and Molecular Dynamic Simulations

“…In order to obtain information on the hidden dynamics behind the native structure, we employed pressure because it enables access to these hidden states by shifting the conformational equilibrium [21]. Furthermore, among various denaturation experiments, analyses of pressure-induced conformational transitions provided distinct information, such as changes in molar volumes and conformational fluctuations upon transition at the amino acid residue level [22,23]. We applied pressure to induce structural transitions in wild-type and D76N β 2 ms and analyzed the changes induced using NMR and MD simulations to clarify the effects of the mutation at the atomic level.…”

Direct Analysis of Mitochondrial Damage Caused by Misfolded/Destabilized Proteins

“…In particular, our study aims to examine whether a protein (any protein) becomes toxic upon aggregation/ destabilization. We chose DENV3-ED3 as a model protein for a practical reason: Several variants with single or few mutations that exhibited uniquely different biophysical properties in terms of oligomerization and thermal stability [ 15 , 16 , 17 ] were available, and DENV3-ED3′s native structure was well characterized [ 18 ]. Altogether, this study provided insights into the mechanisms of cellular and in particular mitochondrial damage caused by non-amyloid-misfolded proteins.…”