Docking and free energy simulations to predict conformational domains involved in hCG–LH receptor interactions using recombinant antibodies

Abstract

Single chain fragment variables (ScFvs) have been extensively employed in studying the protein-protein interactions. ScFvs derived from phage display libraries have an additional advantage of being generated against a native antigen, circumventing loss of information on conformational epitopes. In the present study, an attempt has been made to elucidate human chorionic gonadotropin (hCG)-luteinizing hormone (LH) receptor interactions by using a neutral and two inhibitory ScFvs against hCG. The objective was to dock a computationally derived model of these ScFvs onto the crystal structure of hCG and understand the differential roles of the mapped epitopes in hCG-LH receptor interactions. An anti-hCG ScFv, whose epitope was mapped previously using biochemical tools, served as the positive control for assessing the quality of docking analysis. To evaluate the role of specific side chains at the hCG-ScFv interface, binding free energy as well as residue interaction energies of complexes in solution were calculated using molecular mechanics Poisson-Boltzmann/surface area method after performing the molecular dynamic simulations on the selected hCG-ScFv models and validated using biochemical and SPR analysis. The robustness of these calculations was demonstrated by comparing the theoretically determined binding energies with the experimentally obtained kinetic parameters for hCG-ScFv complexes. Superimposition of hCG-ScFv model onto a model of hCG complexed with the 51-266 residues of LH receptor revealed importance of the residues previously thought to be unimportant for hormone binding and response. This analysis provides an alternate tool for understanding the structure-function analysis of ligand-receptor interactions.

A novel Monoclonal Antibody against Notch1 Targets Leukemia-associated Mutant Notch1 and Depletes Therapy Resistant Cancer Stem Cells in Solid Tumors

“…The wild type and mutant (L1594P) Notch1 NRR proteins were dissolved in 50 mM sodium acetate buffer, pH 5.5, and immobilized on an EDC/NHS[N-ethyl-N’-(3-dimethyl amino propyl) – carbo-di-imide-hydrochloride/ (N-hydroxy succinimide)] -activated CM5 sensor chip as recommended by the manufacturer (BIAcore AB) yielding a surface density of approximately 1000 resonance units. MAbs 604.107, 604.132 and 604.164 were diluted in 10 mM HEPES, 0.15 M NaCl, 3.4 mM EDTA, 0.05% surfactant P-20 binding buffer, pH 7.4 (HBS) and allowed to bind at 25 °C using a flow-rate of 10 μl/min as previously described 27 . The dissociation constant, K D , was calculated from the ratio of dissociation rate (K OFF ) to association rate (K ON ) from three sensorgrams for analyte concentration ranging from 5–10 μM using the curve-fitting BIAevaluation software, version 3.0 (BIAcore AB) and the 1:1 Langmuir model.…”

Exploring PHD Fingers and H3K4me0 Interactions with Molecular Dynamics Simulations and Binding Free Energy Calculations: AIRE-PHD1, a Comparative Study

“…Entropy calculations require a full sampling of the free energy landscape, an extremely computationally demanding step, which can result in unreliable results [36] with standard errors usually with an order of magnitude larger than those associated with the other energetic terms [49]. In addition, the normal mode analysis estimation is often extremely qualitative [50] and the configuration entropy estimate on a short dynamic time range can be non-significant [51]. Based on these considerations and on the fact that we were mainly interested in ΔΔG comp (see further) we decided to neglect the entropic term in our calculations.…”

Insights into differential modulation of receptor function by hinge region using novel agonistic lutropin receptor and inverse agonistic thyrotropin receptor antibodies

“…MAbs against the TSHR-HinR protein were developed according to protocol discussed previously [7]. ScFvs against LHR-HinR protein were selected from Phage human ScFv library (Tomlinson's I + J library) as described previously [8]. Soluble ScFvs were purified by Protein A affinity chromatography.…”