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. 2015 Oct 31;4:375-385.
doi: 10.1016/j.bbrep.2015.10.014. eCollection 2015 Dec.

Structure-based pKa Prediction Provides a Thermodynamic Basis for the Role of Histidines in pH-induced Conformational Transitions in Dengue Virus

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Free PMC article

Structure-based pKa Prediction Provides a Thermodynamic Basis for the Role of Histidines in pH-induced Conformational Transitions in Dengue Virus

Sidhartha Chaudhury et al. Biochem Biophys Rep. .
Free PMC article

Abstract

pH-induced conformational changes in dengue virus (DENV) are critical to its ability to infect host cells. The envelope protein heterodimers that make up the viral envelope shift from a dimer to a trimer conformation at low-pH during membrane fusion. Previous studies have suggested that the ionization of histidine residues at low-pH is central to this pH-induced conformational change. We sought out to use molecular modeling with structure-based pKa prediction to provide a quantitative basis for the role of histidines in pH-induced conformational changes and identify which histidine residues were primarily responsible for this transition. We combined existing crystallographic and cryo-electron microscopy data to construct templates of the dimer and trimer conformations for the mature and immature virus. We then generated homology models for the four DENV serotypes and carried out structure-based pKa prediction using Rosetta. Our results showed that the pKa values of a subset of conserved histidines in DENV successfully capture the thermodynamics necessary to drive pH-induced conformational changes during fusion. Here, we identified the structural determinants underlying these pKa values and compare our findings with previous experimental results.

Keywords: Flavivirus; Histidine; Viral fusion; pKa shift.

Figures

Fig. 1
Fig. 1
Thermodynamic cycle of pH dependence of DENV E-PrM. Thermodynamic cycle of the pH dependence of the unfolded state (U) and a folded state (S) of dengue virus in the fixed uncharged (subscript 0) state and ionizable (subscript i) state. The folded state (S) can refer to any folded conformation, including the immature dimer, mature dimer, and postfusion trimer states.
Fig. 2
Fig. 2
Template structures for DENV E-PrM conformational states. Template structures for the trimer (right) and dimer configurations for mature (bottom left) and immature (top left) DENV. Domains I, II and III of E are shown in cyan, purple, and magenta and PrM is shown in orange. The soluble portion of PrM and E are shown as surfaces, the peri- and trans-membrane helices of PrM and E are shown as cartoons.
Fig. 3
Fig. 3
Structure-based pKa predictions for DENV E-PrM. Calculated pKa values for conserved histidine residues in the immature dimer (blue), mature dimer (purple), and postfusion trimer (red) states using Rosetta-pKa. The ideal pKa value for histidine was set to 6.3, and the maximal allowable pKa range was set between 3.3 and 9.3. Each residue has four pKa values to reflect the pKa value calculated for DENV-1 through DENV-4, respectively.
Fig. 4
Fig. 4
Changes in folding free energy as a function of pH. Contribution of histidine to changes in folding energy as a function of pH (GpHHis) based on the predicted pKa values for conserved histidines for the immature dimer (blue), mature dimer (purple), and postfusion trimer (red) for DENV-1 through DENV-4.
Fig. 5
Fig. 5
Individual residue contributions to protein stability as a function of pH. Individual residue contributions to protein stability at low-pH and neutral pH for the immature dimer, mature dimer, and postfusion trimer shown as by bar graphs (top) and graphical representation (bottom). The various stages and respective environmental pH of the viral life cycle are shown as viral maturation [in the trans-Golgi network (TGN)], viral release into the extracellular environment, and host cell invasion via the endosome. Colors correspond to the energetic contribution to stability with red as destabilizing, gray as neutral, and blue as stabilizing. Structures of E are colored dark gray (PrM), magenta (fusion loops), and salmon (conserved histidine residues).
Fig. 6
Fig. 6
Local environment of H144 and H244 in DENV E-PrM. The local environment around conserved residues H144 (A) and H244 (B), in the immature dimer (left), mature dimer (middle), and postfusion trimer (right). Median pKa values are shown in parentheses. Pr is shown in orange, and E is shown in slate and magenta. Salt bridges between positive and negatively charged residues are shown as dotted lines. Hydrophobic residue side-chains are shown as spheres.
Fig. 7
Fig. 7
Local environment of H98 (PrM), H209, and H261 in DENV E-PrM. The local environment around conserved residues H98 (PrM), H209, and H261, as well as neighboring H27 and H244 in the immature (left) and mature (right) dimer. Median pKa values are shown in parentheses. The coloring and format is identical to Fig. 6.

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