Theoretically engineered DARPins targetting DENV-2 envelope protein / Chong Wei Lim

Chong , Wei Lim (2021) Theoretically engineered DARPins targetting DENV-2 envelope protein / Chong Wei Lim. PhD thesis, Universiti Malaya.

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Abstract

Infectious diseases caused by dengue virus (DENV) have been threatening human health worldwide particularly tropical and sub-tropical regions. Infected population, as reported by WHO, reach 390 million yearly and the global incidence of dengue has tremendously grown. Specific drugs for treating dengue are under development while several antibodies bound to domain III (DIII) envelope (E) protein of DENV were found to prevent the viral entry process. In this study, the potential of Designed Ankyrin Repeat Proteins (DARPins), one of the non-immunoglobulin protein scaffolds, mimicking the binding interactions of antibodies and DIII of DENV envelope (E) protein was explored. Selected DARPins retrieved from the Protein Data Bank were docked to the epitope of domain III as recognised by antibodies under the HADDOCK web server. Representative docked complexes then underwent molecular dynamics simulations (MDs) with AMBER forcefield ff14SB to study their molecular properties. Binding affinity of DARPins to DIIIs was elucidated by free energy calculation using Molecular Mechanics–Poisson- Boltzmann Surface Area/Generalized Born Surface Area (MM-PPSA/GBSA) protocols while the important residues for protein-protein interactions were identified by further decomposing the binding free energy per residue basis. DARPin residues found with unfavourable energy within the binding vicinity then underwent computational site direct mutagenesis. Improved and engineered DARPins in complexed with DIII were simulated under the same parameters for free energy calculations. Gaussian Network Model (GNM), an elastic network model, was then used to investigate the dynamics of the proteins in terms of global mode shape and their dynamic cross-correlations. Global mode shape was observed corresponding to binding free energy, in which a low binding free energy was accompanied by a lower mode shape, or lower mobility. The simulated techniques provide valuable tools in understanding the structural dynamics and energy contribution in designing the DARPins in their binding to the E protein of DENV-2. The protein-protein complexes with different binding activities can clearly be identified by evaluating the binding free energy and global mode shape of the structures from the long timescale MDs.

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