Abstract
This study aimed to design and functionally characterize peptide mimetics of the nanobody (Nb) related to the β2-adrenergic receptor (β2-AR) (nanobody-derived peptide, NDP). We postulated that the computationally derived and optimized complementarity-determining region 3 (CDR3) of Nb is sufficient for its interaction with receptor. Sequence-related Nb-families preferring the agonist-bound active conformation of β2-AR were analysed using the informational spectrum method (ISM) and β2-AR:NDP complexes studied using protein-peptide docking and molecular dynamics (MD) simulations in conjunction with metadynamics calculations of free energy binding. The selected NDP of Nb71, designated P3, was 17 amino acids long and included CDR3. Metadynamics calculations yielded a binding free energy for the β2-AR:P3 complex of ΔG = (−7.23 ± 0.04) kcal/mol, or a Kd of (7.9 ± 0.5) μM, for T = 310 K. In vitro circular dichroism (CD) spectropolarimetry and microscale thermophoresis (MST) data provided additional evidence for P3 interaction with agonist-activated β2-AR, which displayed ~10-fold higher affinity for P3 than the unstimulated receptor (MST-derived EC50 of 3.57 µM vs. 58.22 µM), while its ability to inhibit the agonist-induced interaction of β2-AR with β-arrestin 2 was less evident. In summary, theoretical and experimental evidence indicated that P3 preferentially binds agonist-activated β2-AR.
Highlights
Nanobodies (Nbs) are recombinant, antigen-specific, single-domain, variable fragments of camelid heavy-chain-only antibodies with a broad range of diagnostic, therapeutic and research applications[1], including studies of G-protein-coupled receptors (GPCRs)
One selected computationally characterized Nb-derived peptides (NDPs) was experimentally tested (i) by assessing its ability to bind β2-adrenergic receptor (β2-AR) using techniques for studying protein-protein interactions, i.e., circular dichroism (CD) spectroscopy to detect changes in the conformation of interacting proteins[24] and microscale thermophoresis (MST), a powerful analytical technique for characterizing biomolecular interactions based on the movement of molecules in microscopic temperature gradients; and (ii) by interfering with its function using the previously developed bioluminescence resonance energy transfer (BRET)-based β-arrestin 2 recruitment assay[26]
Based on complementarity-determining region 3 (CDR3) conservation, β2-AR Nbs were classified into four distinct families [A, B, C, and miscellaneous (MISC)]
Summary
Nanobodies (Nbs) are recombinant, antigen-specific, single-domain, variable fragments of camelid heavy-chain-only antibodies with a broad range of diagnostic, therapeutic and research applications[1], including studies of G-protein-coupled receptors (GPCRs). The sole use of flexible ligand docking is not www.nature.com/scientificreports the most reliable method to estimate complex stability and ligand binding affinity; the obtained complexes were subjected to MD simulations and free energy calculations.
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