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

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.

• Language: English
• Keywords: 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
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: VF - Veterinary Faculty
• Publication status: Published
• Publication version: Version of Record
• Year: 2024
• Number of pages: 12 str.
• Numbering: Vol. 14, iss. 4, art. 423
• PID: 20.500.12556/RUL-164912
• UDC: 577
• ISSN on article: 2218-273X
• DOI: 10.3390/biom14040423
• COBISS.SI-ID: 191281155
• Publication date in RUL: 15.11.2024

Record is a part of a journal

• Title: Biomolecules
• Shortened title: Biomolecules
• Publisher: MDPI
• ISSN: 2218-273X
• COBISS.SI-ID: 519952921

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.

Secondary language

• Language: Slovenian
• Keywords: bioinformatika, peptidi, nanotelesa, molekularno modeliranje, β2-adrenergični receptor, in vitro testi