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), Glišić, Sanja (avtor), Kubale, Valentina (avtor), Cotman, Marko (avtor), Mavri, Janez (avtor), Vrecl, Milka (avtor)

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.

• Jezik: Angleški jezik
• Ključne besede: 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
• Vrsta gradiva: Članek v reviji
• Tipologija: 1.01 - Izvirni znanstveni članek
• Organizacija: VF - Veterinarska fakulteta
• Status publikacije: Objavljeno
• Različica publikacije: Objavljena publikacija
• Leto izida: 2024
• Št. strani: 12 str.
• Številčenje: Vol. 14, iss. 4, art. 423
• PID: 20.500.12556/RUL-164912
• UDK: 577
• ISSN pri članku: 2218-273X
• DOI: 10.3390/biom14040423
• COBISS.SI-ID: 191281155
• Datum objave v RUL: 15.11.2024

Gradivo je del revije

• Naslov: Biomolecules
• Skrajšan naslov: Biomolecules
• Založnik: MDPI
• ISSN: 2218-273X
• COBISS.SI-ID: 519952921

Licence

• Licenca: CC BY 4.0, Creative Commons Priznanje avtorstva 4.0 Mednarodna
• Povezava: http://creativecommons.org/licenses/by/4.0/deed.sl
• Opis: To je standardna licenca Creative Commons, ki daje uporabnikom največ možnosti za nadaljnjo uporabo dela, pri čemer morajo navesti avtorja.

Sekundarni jezik

• Jezik: Slovenski jezik
• Ključne besede: bioinformatika, peptidi, nanotelesa, molekularno modeliranje, β2-adrenergični receptor, in vitro testi