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)Sencanski, Milan (Avtor) Glisić, Sanja (Avtor) Kubale, Valentina (Avtor) Cotman, Marko (Avtor) Mavri, Janez (Avtor) Vrecl, Milka (Avtor) bioinformaticsnanobody-derived peptidescomplementary-determining region 2molecular modelingβ2-adrenergic receptorcell-based in vitro assayscomputational biologypeptidesadrenergic receptorsin vitro techniquesThis 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.20242024-11-15 13:47:16Članek v reviji164912sl