Affinity enhancement of nanobody binding to EGFR: in silico site-directed mutagenesis and molecular dynamics simulation approaches

Abstract

Epidermal growth factor receptor (EGFR), a transmembrane glycoprotein, is overexpressed in many cancers such as head-neck, breast, prostate, and skin cancers for this reason it is a good target in cancer therapy and diagnosis. In nanobody-based cancer diagnosis and treatment, nanobodies with high affinity toward receptor (e.g. EGFR) results in effective treatment or diagnosis of cancer. In this regard, the main aim of this study is to develop a method based on molecular dynamic (MD) simulations for designing of 7D12 based nanobody with high affinity compared with wild-type nanobody. By surveying electrostatic and desolvation interactions between different residues of 7D12 and EGFR, the critical residues of 7D12 that play the main role in the binding of 7D12 to EGFR were elucidated and based on these residues, five logical variants were designed. Following the 50 ns MD simulations, pull and umbrella sampling simulation were performed for 7D12 and all its variants in complex with EGFR. Binding free energy of 7D12 (and all its variants) with EGFR was obtained by weighted histogram analysis method. According to binding free energy results, GLY101 to GLU mutation showed the highest binding affinity but this variant is unstable after 50 ns MD simulations. ALA100 to GLU mutation shows suitable binding enhancement with acceptable structural stability. Suitable position and orientation of GLU in residue 100 of 7D12 against related amino acids of EGFR formed some extra hydrogen and electrostatic interactions which resulted in binding enhancement.

Effects of unsaturated fatty acids (Arachidonic/Oleic Acids) on stability and structural properties of Calprotectin using molecular docking and molecular dynamics simulation approach

“…The Nose-Hoover thermostat constant was utilized for fixing the temperature of the system at 310 K. To maintain the pressure of the system at fixed 1bar pressure, the Parrinello-Rahman pressure coupling method was used [28]. The electrostatic interactions were measured using the Particle Mesh Ewald (PME) method with 1.0 nm short-range electrostatic and van der Waals cutoffs [29,30]. Finally, for each complex, the MD simulation process was repeated twice for about 100 ns with time steps of 2 fs on equilibrated systems respectively.…”

In silico assessment of the inhibitory effect of four flavonoids (Chrysin, Naringin, Quercetin, Kaempferol) on tyrosinase activity using the MD simulation approach

“…The Nose-Hoover thermostat constant was used to fix the temperature of the system at 300 K. To maintain the pressure of the system at a fixed 1 bar pressure, the Parrinello-Rahman pressure coupling method was used (Akya et al, 2019). The electrostatic interactions were calculated using the Particle Mesh Ewald (PME) method with 1.0 nm shortrange electrostatic and van der Waals cutoffs (Farasat et al, 2017;Ochoa et al, 2018). Consequently, the process of 50 ns MD simulation for each complex of the enzyme-substrate/inhibitor was performed with time steps of 2 fs on the equilibrated systems.…”

“…Finally, the PMF pattern was provided by the Weighted Histogram Analysis (WHAM) method, which was carried out by GROMACS as "g_wham" command (Zeng et al, 2016). Moreover, for a better recognition of the MD process, the RMSF (root mean square fluctuation) (Chen et al, 2016;Mahapatra et al, 2018), RMSD (root mean square deviation) (Kaur et al, 2019;Shen et al, 2012), Rg (Radius of gyration) (Anantram et al, 2018;Farasat et al, 2017;Lobanov et al, 2008), the inhibitor and substrate distance from the amino acids of the enzyme active site (Corvo et al, 2013;Kato et al, 2017), and the van der Waals and the electrostatic energy of each system were analyzed using GROMACS tools in the period of simulation (Fried and Boxer, 2017;Schutt et al, 2015). Eventually, the final PDB file of MD simulation was plotted using Pymol software (Stourac et al, 2019).…”

Study of HSA interactions with arachidonic acid using spectroscopic methods revealing molecular dynamics of HSA‑AA interactions

“…The system equilibration phase constant substance amount, pressure and temperature (NPT) and constant substance amount, volume and temperature (NVT) as performed at 300 K for 200 psec followed by MD production run for 20 nsec. As described in previous studies (27,28), the atomic coordinates were registered every 2.0 psec during the MD simulation process. In this section parameters, including root mean square deviation (RMSD) and root mean square fluctuation (RMSF), were measured.…”