Affinity enhancement of nanobody binding to EGFR: in silico site-directed mutagenesis and molecular dynamics simulation approaches.
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.
a Department of Medical Biotechnology, Faculty of Medical Sciences , Tarbiat Modares University , Tehran , Iran.
Alireza
A
Farasat
a Department of Medical Biotechnology, Faculty of Medical Sciences , Tarbiat Modares University , Tehran , Iran.
Fatemeh
F
Rahbarizadeh
b Young Researchers and Elite Club, West Tehran Branch , Islamic Azad University , Tehran , Iran.
Ghader
G
Hosseinzadeh
c Faculty of Sciences, Roudehen Branch, Department of Biology , Islamic Azad University , Roudehen , Iran.
Sharareh
S
Sajjadi
d Nanobiotechnology Research Center , Baqiyatallah University of Medical Science , Tehran , Iran.
Mehdi
M
Kamali
e Molecular Biology Research Center , Baqiyatallah University of Medical Science , Tehran , Iran.
Amir Homayoun
AH
Keihan
D020816:Amino Acid Motifs; D000915:Antibody Affinity; D000918:Antibody Specificity; D001665:Binding Sites; D018360:Crystallography, X-Ray; D066246:ErbB Receptors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D061905:Single-Domain Antibodies / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity; D013816:Thermodynamics
D061905:Single-Domain Antibodies; C512478:EGFR protein, human; D066246:ErbB Receptors
EGFR; affinity enhancement; molecular dynamic; nanobody 7D12