PREDICTING THE BINDING PROPERTIES OF THE SALIVARY RHODNIUS NEGLECTUS ODORANT-BINDING PROTEIN USING MOLECULAR DOCKING, EXPERIMENTAL AND BIOPHYSICAL ASSAYS

Gabriel dos Santos Silva; Paula  Beatriz Santiago; Eric Faudry; Aisel Valle; Giulia Causin Vieira; Kaio Luís da Silva Bentes; Flavia Nader; Sebastien Oliver Charneau; Izabela Marques Dourado Bastos Charneau; Jaime Martins de Santana; Melina Mottin; Carla Nunes de Araújo

Paper Title

Authors

Gabriel dos Santos Silva
Paula Beatriz Santiago
Eric Faudry
Aisel Valle
Giulia Causin Vieira
Kaio Luís da Silva Bentes
Flavia Nader
Sebastien Oliver Charneau
Izabela Marques Dourado Bastos Charneau
Jaime Martins de Santana
Melina Mottin
Carla Nunes de Araújo

Modality

Poster

Subject area

Proteins and Proteomics

Publishing Date

08/11/2023

Country of Publishing

Brazil | Brasil

Language of Publishing

Inglês

Paper Page

https://www.even3.com.br/anais/xmeeting2023/638543-predicting-the-binding-properties-of-the-salivary-rhodnius-neglectus-odorant-binding-protein-using-molecular-dock

ISBN

978-65-272-0061-1

Keywords

Hematophagy, Rhodnius neglectus, Saliva, OBP, Molecular Dynamics, Ensemble docking.

Summary

Rhodnius neglectus is a triatomine insect from the Reduviidae family that presents a variety of pharmacologically bioactive substances in its saliva to counteract hemostasis, inflammation, and modulate the host's immune response. If infected by Trypanosoma cruzi, it can become a vector for Chagas disease and transmit the parasite by contaminating wounds caused by feeding on vertebrate hosts with its feces. Salivary odorant-binding protein (OBP) from the D7 protein family may play a critical role in inhibiting host responses, as certain members capture biogenic amines and eicosanoids that are immune response and coagulation agonists in vertebrates. In a prior study, we identified an OBP in R. neglectus sialome. Our aim is to investigate and characterize a R. neglectus OBP (RnOBP) member and identify potential inhibitors using in silico and experimental methods. To model the 3D structure, and study its binding sites and conformational behavior, we performed three-dimensional structure prediction, binding site prediction, and molecular dynamics simulations. We used ensemble docking to select promising ligands from a small library of molecules related to D7 proteins OBP. The Alphafold server was used to obtain the 3D RnOBP model by artificial intelligence. The model was evaluated by the Molprobity server for statistical quality. The CASTp server was used to identify protein binding sites. Molecular dynamic simulations were performed by the NAMD program and revealed the mobile and rigid regions of the protein and its main conformations during the MD trajectory. Ensemble docking calculations prioritized some molecules such as epinephrine and prostaglandin analogs (U46619) from the clustered protein conformations identified. In the future, a virtual screening campaign of a commercial dataset of molecules will be conducted to identify promising new RnOBP inhibitors. Furthermore, the coding sequence of OBP was cloned into pET100/D-TOPO, and the heterologous protein was produced by Rosetta(DE3)pLysS cells. Bacterial cell extracts were obtained by sonication in lysis buffer. After centrifugation, the insoluble fraction was purified using the HisTrap FF nickel column. The fractions containing the protein were dialyzed and refolded. The expression in Rosetta(DE3)pLysS cells showed a great insoluble fraction and a small soluble fraction. The dialyzed and refolded insoluble fraction purification gave a yield of 754 ug/L of bacterial culture. The sample was then submitted to Size Exclusion Chromatography (SEC) and Analytical Ultracentrifugation (AUC) to confirm the structural information of RnOBP. The current investigation focuses on circular dichroism, fluorimetry, coagulation test, MALDI-TOF analysis, and isothermal titration calorimetry to determine the protein's structure and viability, and to characterize its interactions experimentally, thereby establishing its function in the bite environment.

Title of the Event: X-Meeting / BSB 2023
City of the Event: Curitiba
Title of the Proceedings of the event: X-Meeting presentations
Name of the Publisher: Even3
Means of Dissemination: Meio Digital

How to cite

SILVA, Gabriel dos Santos et al.. PREDICTING THE BINDING PROPERTIES OF THE SALIVARY RHODNIUS NEGLECTUS ODORANT-BINDING PROTEIN USING MOLECULAR DOCKING, EXPERIMENTAL AND BIOPHYSICAL ASSAYS.. In: X-Meeting presentations. Anais...Curitiba(PR) Campus da indústria, 2023. Available in: https//www.even3.com.br/anais/xmeeting2023/638543-PREDICTING-THE-BINDING-PROPERTIES-OF-THE-SALIVARY-RHODNIUS-NEGLECTUS-ODORANT-BINDING-PROTEIN-USING-MOLECULAR-DOCK. Access in: 02/08/2025