20.500.12556/RUL-164912 Computational modeling and characterization of peptides derived from nanobody complementary-determining region 2 (CDR2) targeting active-state conformation of the β$_2$-adrenergic receptor (β$_2$AR) This study assessed the suitability of the complementarity-determining region 2 (CDR2) of the nanobody (Nb) as a template for the derivation of nanobody-derived peptides (NDPs) targeting active-state β$_2$-adrenergic receptor (β$_2$AR) conformation. Sequences of conformationally selective Nbs favoring the agonist-occupied β$_2$AR were initially analyzed by the informational spectrum method (ISM). The derived NDPs in complex with β$_2$AR were subjected to protein–peptide docking, molecular dynamics (MD) simulations, and metadynamics-based free-energy binding calculations. Computational analyses identified a 25-amino-acid-long CDR2-NDP of Nb71, designated P4, which exhibited the following binding free-energy for the formation of the β$_2$AR:P4 complex (ΔG = −6.8 ± 0.8 kcal/mol or a Ki = 16.5 μM at 310 K) and mapped the β$_2$AR:P4 amino acid interaction network. In vitro characterization showed that P4 (i) can cross the plasma membrane, (ii) reduces the maximum isoproterenol-induced cAMP level by approximately 40% and the isoproterenol potency by up to 20-fold at micromolar concentration, (iii) has a very low affinity to interact with unstimulated β$_2$AR in the cAMP assay, and (iv) cannot reduce the efficacy and potency of the isoproterenol-mediated β$_2$AR/β-arrestin-2 interaction in the BRET$^2$-based recruitment assay. In summary, the CDR2-NDP, P4, binds preferentially to agonist-activated β$_2$AR and disrupts Gαs-mediated signaling. bioinformatics nanobody-derived peptides complementary-determining region 2 molecular modeling β2-adrenergic receptor cell-based in vitro assays computational biology peptides adrenergic receptors in vitro techniques bioinformatika peptidi nanotelesa molekularno modeliranje β2-adrenergični receptor in vitro testi true false true Angleški jezik Slovenski jezik Članek v reviji 2024-11-15 13:47:16 2024-11-15 13:47:23 2024-11-27 11:40:51 0000-00-00 00:00:00 2024 0 0 12 str. iss. 4, art. 423 Vol. 14 2024 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 577 2218-273X 10.3390/biom14040423 191281155 biomolecules-14-00423-v2.pdf biomolecules-14-00423-v2.pdf 1 27318EC81720E6751E6CFC71D21AAF3E 7e53d0c81386a07cd0e4bc686cfb383d6dfb6be100adfd952da41458ab7c67c1 c7cea93e-a34f-11ef-b232-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=195763 https://www.mdpi.com/2218-273X/14/4/423 1 64b7f0da-a34f-11ef-b232-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=195762 Veterinarska fakulteta 0 0 0